F3 Microbes and biotechnology F.3.1 – F 3.5

F. 3.1 State that reverse transcriptase catalyzes the production of RNA to DNA

Reverse transcriptase is an enzyme isolated from retroviruses which catalyses the production of DNA from an RNA template

RNA as Viral Genetic Material The broadest variety of RNA genomes is found in viruses that infect animals Retroviruses use reverse transcriptase to copy their RNA genome into DNA HIV (human immunodeficiency virus) is the retrovirus that causes AIDS (acquired immunodeficiency syndrome) © 2011 Pearson Education, Inc.

Figure 19.8 Membrane of white blood cell Glycoprotein Viral envelope HIV Capsid RNA (two identical strands) HOST CELL Reverse transcriptase HIV Reverse transcriptase Viral RNA RNA-DNA hybrid DNA 0.25 m HIV entering a cell NUCLEUS Provirus Chromosomal DNA RNA genome for the next viral generation Figure 19.8 The replicative cycle of HIV, the retrovirus that causes AIDS. mRNA New virus New HIV leaving a cell

RNA (two identical strands) Reverse transcriptase HOST CELL Figure 19.8a Glycoprotein Viral envelope Capsid RNA (two identical strands) Reverse transcriptase HOST CELL HIV Reverse transcriptase Viral RNA RNA-DNA hybrid DNA NUCLEUS Chromosomal DNA Provirus RNA genome for the next viral generation Figure 19.8 The replicative cycle of HIV, the retrovirus that causes AIDS. mRNA New virus

Membrane of white blood cell Figure 19.8b Membrane of white blood cell HIV Figure 19.8 The replicative cycle of HIV, the retrovirus that causes AIDS. 0.25 m HIV entering a cell New HIV leaving a cell

The viral DNA that is integrated into the host genome is called a provirus Unlike a prophage, a provirus remains a permanent resident of the host cell The host’s RNA polymerase transcribes the proviral DNA into RNA molecules The RNA molecules function both as mRNA for synthesis of viral proteins and as genomes for new virus particles released from the cell © 2011 Pearson Education, Inc.

Animation: HIV Reproductive Cycle Right-click slide / select “Play” © 2011 Pearson Education, Inc.

F. 3.2 Explain how reverse transcriptase is used in molecular biology

A complementary DNA (cDNA) library is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell A cDNA library represents only part of the genome— only the subset of genes transcribed into mRNA in the original cells © 2011 Pearson Education, Inc.

Reverse transcriptase is used as part of recombinant DNA technology to produce genes for gene transfer The enzyme catalyses the production of a complementary (single) strand of copy DNA (cDNA) from an mRNA template As RNA undergoes post-transcriptional modification (i.e. splicing) prior to forming mRNA, cDNA does not contain introns As bacteria lack the machinery for intron removal, genes spliced into bacterial hosts need introns removed in order to generate functional proteins Examples of how reverse transcriptase has been used in molecular biology include: The mass production of human insulin by E. coli cells The generation of cDNA libraries for use in DNA microarrays (DNA fingerprinting)

DNA in nucleus mRNAs in cytoplasm Figure 20.6-1 Figure 20.6 Making complementary DNA (cDNA) from eukaryotic genes.

Reverse transcriptase Poly-A tail mRNA 5 3 3 5 DNA strand Primer Figure 20.6-2 DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail mRNA 5 A A A A A A 3 3 T T T T T 5 DNA strand Primer Figure 20.6 Making complementary DNA (cDNA) from eukaryotic genes.

Reverse transcriptase Poly-A tail mRNA 5 3 3 5 DNA strand Primer Figure 20.6-3 DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail mRNA 5 A A A A A A 3 3 T T T T T 5 DNA strand Primer 5 A A A 3 A A A 3 T T T T T 5 Figure 20.6 Making complementary DNA (cDNA) from eukaryotic genes.

Reverse transcriptase Poly-A tail mRNA 5 3 3 5 DNA strand Primer Figure 20.6-4 DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail mRNA 5 A A A A A A 3 3 T T T T T 5 DNA strand Primer 5 A A A 3 A A A 3 T T T T T 5 Figure 20.6 Making complementary DNA (cDNA) from eukaryotic genes. 5 3 3 5 DNA polymerase

Reverse transcriptase Poly-A tail mRNA 5 3 3 5 DNA strand Primer Figure 20.6-5 DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail mRNA 5 A A A A A A 3 3 T T T T T 5 DNA strand Primer 5 A A A 3 A A A 3 T T T T T 5 Figure 20.6 Making complementary DNA (cDNA) from eukaryotic genes. 5 3 3 5 DNA polymerase 5 3 3 5 cDNA

F.3.3 Distinguish between somatic and germ line therapy

Gene therapy is the insertion of genes into an individual's cells and tissues in order to treat genetic diseases

F.3.4 Outline the use of viral vectors in gene therapy http://learn.genetics.utah.edu/content/genetherapy/gtintro/ What is gene therapy?

Concept 20.4: The practical applications of DNA technology affect our lives in many ways Many fields benefit from DNA technology and genetic engineering © 2011 Pearson Education, Inc.

Human Gene Therapy Gene therapy is the alteration of an afflicted individual’s genes Gene therapy holds great potential for treating disorders traceable to a single defective gene Vectors are used for delivery of genes into specific types of cells, for example bone marrow Gene therapy provokes both technical and ethical questions © 2011 Pearson Education, Inc.

Insert RNA version of normal allele into retrovirus. Figure 20.23 Cloned gene 1 Insert RNA version of normal allele into retrovirus. Viral RNA 2 Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Retrovirus capsid 3 Viral DNA carrying the normal allele inserts into chromosome. Bone marrow cell from patient Figure 20.23 Gene therapy using a retroviral vector. Bone marrow 4 Inject engineered cells into patient.

Viral vectors have been used to facilitate the replacement of defective genes with healthy, functional copies Individuals with severe combined immunodeficiency (SCID) may have be unable to synthesize the enzyme adenosine deaminase (ADA) White blood cells or bone marrow cells are removed and, using a viral vector, a copy of the normal gene is integrated into the cell's genome When the cells are replaced in the body of the patient the normal gene is expressed, resulting in the production of ADA and the treatment of SCID There are still technical problems to be solved before this becomes viable technology – e.g. ensuring correct amount at right time and place

F.3.5 Discuss the risks of gene therapy

Safety and Ethical Questions Raised by DNA Technology Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology © 2011 Pearson Education, Inc.

Most public concern about possible hazards centers on genetically modified (GM) organisms used as food Some are concerned about the creation of “super weeds” from the transfer of genes from GM crops to their wild relatives Other worries include the possibility that transgenic protein products might cause allergic reactions © 2011 Pearson Education, Inc.

As biotechnology continues to change, so does its use in agriculture, industry, and medicine National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology © 2011 Pearson Education, Inc.

Risks associated with Gene Therapy Undesirable health effects (e.g. cancers / death) If gene insertion occurs in the wrong location it may affect the functioning of pre-existing genes that are vital within the genome Viral vectors may infect healthy cells or tissues Virus may revert to original form (mutate) and become pathogenic Virus entry may trigger an immune response leading to inflammation, toxicity and organ failure Treatment must be repeated at regular intervals, increasing likelihood of adverse treatment response with time