DNA Recombination

10. What is an âinsertion elementâ vs. a âsimple transposonâ vs. a âretrotransposonâ?

11. What enzymes are involved in transposition and what do they do?

12. What are direct repeats vs. inverted repeats vs. long terminal repeats? Which ones actually move with the transposon?

13. How can a simple cut and paste transposon increase the size of the genome?

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Class 6: Viruses (and Cells, continued)

Making connections: viruses and microbes Match the cell biology concept (on page 1) with an important application of that concept (on page 2). Then answer the follow-up questions at the bottom of page 2.

Cell Biology concepts
1. Bacteria that cannot construct new cell wall material become fragile and break down.
2. During influenza infection, the viral enzyme neuraminidase is required to release new viruses from the host cell in which they were made. Once released, the new viruses can infect many additional cells.
3. Thermus aquaticus is a bacterium found in hot springs and can live at temperatures up to ~80Â°C (175Â°F). It has adapted so that its enzymes are most functional at the elevated temperatures, compared with many organismsâ enzymes that function best ~37Â°C (98Â°F).
4. Until the isolation and investigation of retroviruses, scientists thought that RNA could be copied from DNA, but DNA could not be copied from RNA. Research on this class of viruses led to an update to this âCentral Dogmaâ of biology.
5. Some bacteria have adapted, so that the enzymes involved in making cell wall materials have a slightly different shape and can no longer be bound by certain drugs.
6. Rhizobia are bacteria that colonize the roots of certain plants (legumes, including beans and peas). The host plant benefits from the prokaryotesâ ability to âfixâ nitrogen, converting N2 from the atmosphere into a form that is usable by the plant.
7. Many species of bacteria contain the enzyme cellulase. More than 10 billion such bacteria and other symbiotic microbes may be found in a single milliliter of material from inside the digestive system of ruminant mammals.
8. Fungi, including those that cause athleteâs foot, use ergosterol to maintain healthy membranes. Animal cells do not make ergosterol, they use a similar lipid â cholesterol.
9. During retrovirus infection, a DNA copy is made of the virusâs RNA genome. A cut is made in the host cellâs DNA and the DNA copy of the viral genome is pasted into the host chromosome by a viral enzyme called integrase.
10. Prokaryotic cells contain smaller ribosomes (â70Sâ ribosomes) than eukaryotic cells (â80Sâ ribosomes). Both types of ribosomes synthesize proteins, but the macromolecules they use to do it are somewhat different.

2
Applications
_______ Oseltamivir, also called Tamiflu, binds and inhibits the viral protein neuraminidase.
_______ Clotrimazole is a drug that is used to treat certain infections. It interferes with synthesis of ergosterol, a lipid involved in phospholipid membranes.
_______ Penicillin is a drug that interferes with synthesis of peptidoglycans (these are biological molecules that are part protein and part carbohydrate) that are present in certain cell walls.
_______ HIV is often treated with several drugs that inhibit different parts of the viral replicative cycle. Raltegravir is an example of a drug that interferes with the integration of viral genes into the host cellâs chromosome.
_______ Cows, like other vertebrates, lack the ability to breakdown cellulose. Yet they are able to live on a diet of grass and other vegetation high in cellulose.
_______ Enzymes isolated from extremophiles have been very useful in laboratory research. For example, Taq polymerase facilitates PCR, a revolutionary technique for production of DNAs of particular sequences, which requires cycling reactions through high temperatures.
_______ Plants and animals require nitrogen to make biological macromolecules, however theyâre unable to convert atmospheric nitrogen (N2) into a biologically useful form.
_______ Azithromycin is a drug that binds to 70S ribosomes and blocks synthesis of any new proteins.
_______ Methicillin-Resistant Staphylococcus aureus (MRSA) can cause life-threatening infections of the skin and bloodstream. One of the reasons MRSA infections are so serious is that they are resistant to treatment by many antibiotics, including methicillin and penicillin.
_______ The enzyme reverse transcriptase, isolated from retroviruses, is an important tool in molecular biology research. Scientists interested in learning what genes are turned on in particular cells use it to make DNA copies of RNAs they are studying (The DNA copies are useful because they are more stable).

When you have completed the matching activity, answer these follow-up questions:
â¢ What types of infections can be treated with the following drugs?
o Penicillin
o Clotrimazole
o Azithromycin
o Tamiflu
o Raltegravir
â¢ Why are these drugs (above) safe for you to take? That is, why donât they kill your cells?
â¢ Cancer cells are your own human cells that have become deregulated so that they divide and pile up in an uncontrolled way. Many chemotherapy drugs treat cancer by interfering with cell division and other cell processes.
o Why are there so many more side effects for individuals undergoing chemotherapy than for individuals taking antibiotics?

HELP WITH BIO MULTI CHOICE QUESTIONS:

QUESTION 2

Which of the following structures discussed in class is NOT found on all bacteria?
Flagella
Capsid
Fimbria
Plasma membrane

QUESTION 3
You are presented with a strain of bacteria that is resistant to the antibiotic penicillin. What steps would you take to confirm that you have a resistant bacterium?
Perform a Gram stain to determine pink stained Gram-positive bacteria.
Perform a Gram stain to determine purple stained Gram-positive bacteria.
Perform a Gram stain to determine pink stained Gram-negative bacteria.
Perform a Gram stain to determine purple stained Gram-negative bacteria.

QUESTION 4
With respect to bacterial growth into a fresh culture tube containing nutrient broth, at which phase do organisms undergo binary fission the most rapidly?
Lag phase
Stationary
Exponential
death

QUESTION 5
How would antibiotic resistance be passed from one bacterium to another bacterium?
Through the flagella via a plasmid.
Through the sex pilus via chromosomal DNA.
Through the sex pilus via a plasmid.
Through the flagella via chromosomal DNA.

QUESTION 6
When an F plasmid integrates into the host chromosome, the strain is referred to as

Hfr.
F'.
F-.
F+.

QUESTION 7
If you remove the peptidoglycan layer from a Gram-positive cell, it would still stain purple with a Gram stain (T/F).
True
False

QUESTION 8
During infection with a bacteriophage, bacterial DNA is packaged into a capsid and will:

be integrated into the genome of a new bacterium upon ligation.
be integrated into the genome of a new virion upon recombination.
be integrated into the genome of a new bacterium upon recombination.
be integrated into the genome of a new virion upon liagation.

QUESTION 9
Which of the following statements best describes most bacteria?
They are generally harmful.
They are limited to living in a few habitats.
They are very common in the environment.
They are responsible for the âcommon cold.â

QUESTION 10
Prokaryotes lack some parts found in eukaryotic cells, including which item?
a nuclear membrane
DNA
one or more chromosomes
a plasma membrane
all of the above

QUESTION 11
With respect to gram negative bacteria, what is the order of the bacterial cell structure from outside to inside?
Thin peptidoglycan layer, plasma membrane

thick peptidoglycan layer, plasma membrane

outer membrane, thick peptidoglycan layer, plasma membrane

outer membrane, thin peptidoglycan layer, plasma membrane

QUESTION 12
In transformation, a piece of DNA from a dead host can be taken up by living host

True
False

Cystic Fibrosis (CF) is an autosomal recessive genetic disease that is characterized by the secretion of thick, abnormal mucous in the lungs. The mutated gene that leads these symptoms is the CF transmembrane conductance regulator (CFTR gene). The encoded protein is a chloride channel that moves chloride ions out of epithelial cells. In a homozygous individual, the lack of this chloride channel function in a mutant slows the flow of water into extracellular spaces. This lack of water export leads to the heavy mucus in the lungs of CF patients. Although children with CF would die in childhood, current treatments provide life expectancies into the 30âs.

CF is the most frequently inherited genetic disorder among American children. It affects 1 of every 2,500 children; 30,000 currently have the disease. Although its frequency is higher in European populations, it affects all human populations. Heterozygous carrier frequencies in the US are estimated to be 1 out of 29 Caucasians, 1 out of 46 Hispanics, 1 out of 61 African Americans and 1 out of 90 Asians. There are genetic screens for carriers of CF but they are not routinely performed despite this high frequency of carriers. At least all states in the US proscribe testing of newborns to identify children with CF early so that treatment can be started. Newborn tests for CF: http://www.cff.org/AboutCF/Testing/NewbornScreening/ScreeningforCF/ . If CF is such a common genetic disease, and such a simple Mendelian trait, why is genetic screening of parents not done more frequently? Is it just economic, a pain to do, or is it more complex such that the results of genetic testing are not just positive or negative?

Answer the following questions:
1. If two parents are heterozygous carriers of CF, show a pedigree with four children showing the incidence of carriers and afflicted children. If each unafflicted child (carrier or not) mates with another person, who has a 1 out of 30 chance of being a carrier of CF, what is the expected incidence CF disease in the third generation (frequency of CF in any one of grandchildren of the original couple)?

2. Pleiotropy of Cystic Fibrosis. Watch the two videos on the left at:

http://www.cftrscience.com/MOD-animation
or, if you can not view these, read the short NIH Genetics Home Reference for Cystic Fibrosis http://ghr.nlm.nih.gov/condition/cystic-fibrosis

Connect the description with one of the following videos, or related ones (there are many) you see:
video (10 minutes) of daily life of a child with Cystic Fibrosis http://www.youtube.com/watch?v=WUsuGKzdDg8
Lauraâs (20 year old) video diary of life with CF http://www.youtube.com/watch?v=Pgrf8qJXhpc&feature=related

In your own words, describe the range of CF phenotypes. List the other organs besides lungs that are affected and how does it affects those organs.

3.The DNA sequence of the CFTR gene is 170,000 base pairs in length and encodes the CFTR protein that is 1480 amino acids long. Open the accompanying text file (in Canvas/Files/Discussion folder) âCFTR gene DNA sequenceâ. You will see an alignment of the NIH human genome sequence of the CFTR gene â called âReferenceâ rather than âwild-typeâ- against a gene sequence containing a common mutation in CFTR. Look at the alignment and determine how the DNA sequence differs between them. What is the position number where they differ? How many and what nucleotides do they differ in? A â-â mark in one sequence in the alignment means that that base sequence is missing the corresponding bases in the other sequence. The sequence is a long one but the differences are in bolded bright red text. Matching sequence is marked with a | linking the two sequences. Look near the sequence AAATATCATC if you use FIND tool in Word.

In another file, you will an alignment of the reference translated into a protein sequence, compared to the mutant allele translated into protein sequence. The differences are again in red whereas the areas where the proteins match are in black. Look at the alignment. How do these two proteins differ? Again, find the position number and describe how they differ at that location. A â-â mark in the alignment means that amino acid is missing when compared to the other sequence.

4. Given that the CFTR gene can be analyzed for a mutation that causes CF, couples interested in being tested as carriers face several choices given the results.
What could be a consequence of a false positive result (couple is identified as carriers but they would not produce any children with symptoms)?

What could be a consequence of a false negative result (a test indicates the couple is not both carriers but they would produce some children with CF)?

5. The sensitivity of the test can vary with ethnic group. The following is a chart from the NCBI Genetic testing site. A sensitivity less than 100% would be likely to produce false negative results from an inability to determine that someone is carrier.

CF Carrier Test Sensitivity by Ethnic Group http://www.ncbi.nlm.nih.gov/projects/GeneTests/static/concepts/primer/ptscarr5.shtml
Ethnic Group ¹	Population Frequency of CF	Sensitivity of Carrier Detection ²
Caucasian	1 in 29	90%
Ashkenazi Jewish	1 in 29	97%
Native American	1 in 32	94%
Latino	1 in 46	57%
African-American	1 in 60	75%
Asian	1 in 90	30%