1. F3 Microbes and biotechnology
F.3.1 – F 3.5

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

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

4. 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.

5. 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

6. 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

7. 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

8. 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.

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

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

11. 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.

12. 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)

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

14. Reverse transcriptase Poly-A tail mRNA 5 3 3 5 DNA strand Primer
Figure
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.

15. Reverse transcriptase Poly-A tail mRNA 5 3 3 5 DNA strand Primer
Figure
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.

16. Reverse transcriptase Poly-A tail mRNA 5 3 3 5 DNA strand Primer
Figure
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

17. Reverse transcriptase Poly-A tail mRNA 5 3 3 5 DNA strand Primer
Figure
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

18. F.3.3 Distinguish between somatic and germ line therapy

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

20. F.3.4 Outline the use of viral vectors in gene therapy
What is gene therapy?

21. 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.

22. 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.

23. 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 Gene therapy using a retroviral vector.
Bone marrow 4
Inject engineered cells into patient.

24. 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

26. F.3.5 Discuss the risks of gene therapy

27. 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.

28. 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.

29. 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.

30. 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