Concept 25 Some viruses store genetic information in RNA.

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HI! David Baltimore showed that retroviruses have a gene for reverse transcriptase (RT) called pol, and also carry the RT enzyme itself into cells. In his experiment, Baltimore showed that adding trypsin to the viral particles destroys the reverse transcriptase and no DNA is made. Why didn't the viral particle in the test tube make new reverse transcriptase from the gene? There are no tRNAs or ribosomes. (Yes, tRNAs and ribosomes are needed for translation — making protein from mRNA.) There are no amino acids. (Yes, amino acids are needed to make protein.) Both of the above. (That is correct.) There are no RNA polymerases. (No, don't need RNA polymerase because there is viral RNA.) There are no DNA polymerases. (No, DNA polymerase replicates DNA.) Ribosomes, tRNA, amino acids and RNA must be present to make protein. The RT protein is coded in the viral RNA, but none of the other components is present to translate the RNA. Retroviruses can be used as vectors to deliver new genes to animal cells. First, scientists insert DNA with the gag, pol, and env genes into the DNA of cells in culture. These cells are called producer cells because they produce the viral proteins — gag, pol and env. However, they do not produce viruses. Why not? There are no LTRs. (That is correct.) There is no reverse transcriptase. (Reverse transcriptase is present but not needed.) There is no RNA polymerase. (RNA polymerase is present.) There are no amino acids. (No, amino acids are present.) There are no tRNAs or ribosomes. (No, both are present.) LTRs have several functions. They not only help integrate the viral genome into the DNA of an infected cell, they also indicate where RNA transcription begins and ends. Without LTRs, no viral RNA is produced. Without RNA, there is no virus. The next step in turning a retrovirus into a vector with a new gene is to transform the producer cells with a plasmid that contains the new gene. Once transformed with the plasmid, these cells produce modified virus. What sequences does the virus now carry on its genome? RNA genome contains two LTRs, gag, pol, and env. (That is incorrect.) RNA genome contains gag, pol, and env. (That is incorrect.) RNA genome contains two LTRs and new gene. (That is correct.) DNA genome contains two LTRs and new gene. (No, retroviruses carry RNA.) DNA genome contains gag, pol, and env. (No, retroviruses carry RNA.) Remember that LTRs bracket the sequences that will be made into viral RNA. The producer cell DNA lacks LTRs so the gag, pol, and env genes are not transcribed into viral RNA. LTRs on the plasmid DNA initiate RNA transcription of the new gene and LTRs. Let's suppose there is a genetic disorder where white blood cells do not produce a needed enzyme. How can we use retrovirus as a vector to deliver a new gene to the patient, so that white blood cells would produce the missing enzyme? First, we need to choose the cells for gene insertion. Although white blood cells are the target, they eventually die and any changes made will not be permanent. White blood cells are derived from bone marrow stem cells, which can reproduce themselves and make more white blood cells. Inserting the gene into bone marrow stem cells can make the gene transfer permanent. If the doctor could remove the stem cells from the patient, transform them with the retrovirus, and reintroduce the cells back into the patient, which cells will carry the new gene? all the patient's future cells. (No, the inserted cells do not produce cells for the entire body.) only the reintroduced cells. (No, the reintroduced cells will produce other cells with the gene.) the reintroduced cells, and all future stem and white blood cells.(That is correct.) reintroduced cells and future stem cells. (No, the reintroduced cells produce other cells with the gene.) The reintroduced stem cells carry the gene and will pass it on to new cells they create, including future stem and bone marrow cells. Because the stem cells only make white blood cells, none of the patient's other cells are affected. For technical reasons, scientists have not successfully transformed bone marrow stem cells. However, they have inserted a new gene into a patient's white blood cells, which then expressed the missing enzyme.