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.
-Microarrays are chips that contain hundreds of microscopic wells, each of which can detect a distinct nucleic acid. Prior to exposure to cDNA, the wells of the microarray described in the passage most likely contained:

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.
-Microarrays are chips that contain hundreds of microscopic wells, each of which can detect a distinct nucleic acid. Prior to exposure to cDNA, the wells of the microarray described in the passage most likely contained:

Quizplus · Accepted Answer

11ecba2d_102d_e323_a3bf_c17fca072aa0_MD0008_00 A gene within genomic DNA consists of two strands: a sense strand (coding strand) and an antisense (noncoding) strand. RNA is produced when RNA polymerase uses the antisense strand as a template to make a new nucleic acid. The newly synthesized RNA strand will accordingly have the same sequence as the sense strand of the DNA (with the exception that RNA contains uracil instead of thymine). During the formation of cDNA, the enzyme reverse transcriptase produces a single-stranded DNA molecule using mRNA as a template. The cDNA strand is complementary to the mRNA and therefore is complementary to the sense strand of the genomic DNA (ie, cDNA is an antisense strand). The cDNA in the passage was obtained from zebrafish; therefore, to detect the cDNA, the microarray must contain single-stranded DNA from zebrafish genes. Because cDNA is complementary to the sense strand of genomic DNA, the single-stranded DNA must correspond to (ie, have the same sequence as) the sense strand of zebra fish genes, allowing it to hybridize with cDNA. (Choice A) Double-stranded DNA is already hybridized and would be unable to anneal to the cDNA probe. The cDNA would be washed away from all wells, and no signal would be detected. (Choice B) Proteins perform many different functions within cells, but only a subset (eg, transcription factors) bind to DNA. Accordingly, the cDNA would be unlikely to hybridize with zebrafish proteins, and proteins would not be useful in analyzing gene expression. (Choice C) Antibodies typically bind proteins, not DNA. Antibodies may be used in protein identification techniques such as ELISA but would not hybridize with cDNA. Educational objective: During cDNA synthesis, reverse transcriptase uses mRNA as a template to synthesize a new single-stranded DNA molecule. Because mRNA has the same sequence as the sense strand of genomic DNA (using uracil instead of thymine), cDNA will have the same sequence as the antisense strand. Accordingly, cDNA hybridizes with single-stranded DNA that corresponds to the sense strand of genomic DNA.