Biology 30 - Patricia Schwandt Courses - Home



Biology 30

Protein Synthesis

General Outcome C3: Students will explain classical genetics at the molecular level.

A. The Genetic Code

Polypeptides are long chains of amino acids. As polypeptides grow longer, or are attached together, they are called proteins. The sequence of amino acids in a polypeptide determines the characteristics and nature of the protein. Therefore, by ordering the sequence of amino acids, the DNA controls the kinds of proteins, which are built by the cell.

The language is composed of series of nucleotides to represent information.

DNA has only four kinds of nucleotides, but must code for 20 different amino acids used to build polypeptides. Just as a specific series of dots and dashes is used to represent one letter in Morse code, a specific series of nucleotides is used as the code for each amino acid. This series of nucleotides is called a codon. A gene, which is the “blueprint” for a polypeptide, is a long series of codons. The number of codons in different genes varies depending upon the size of the polypeptide chain to be built.

Proteins are large, complex molecules made up of many amino acids joined together. Proteins can be classified into two common types:

1. Functional Proteins – may be enzymes, antibodies, or hormones. Enzymes are needed to lower the activation energy required for chemical reactions to occur inside the cells of organisms. Without the thousands of specific enzymes life wouldn’t be possible. Antibodies defend the body against foreign substances, and hormones help control many processes in organisms.

2. Structural Proteins – include muscles, parts of the cell membranes, hair, tendons, and ligaments.

How many nucleotides are there in each codon?

If there were only one, there could only be four codes, representing only four different amino acids.

How many different codons can be made if there are two nucleotides in each, for example, AA, AT, AC, AG, and so on? Will this be enough to represent all 20 amino acids?

Since it is only possible to make 16 different codons if two nucleotides are used for each (4x4=16), there must be at least three nucleotides in each codon. This means that there are 64 possible combinations (4x4x4).

A series of biochemical studies in the early 1960’s confirmed this number, and also established the specific nucleotide composition of each codon.

Proteins are found in every living cell. It is used as building blocks for all parts of the cell (membranes, organelles, nucleic acids). Cells are responsible for producing their own proteins. The blueprint for the protein is contained in the nucleus (DNA) and the protein is manufactured in the ribosome.

By linking defective enzymes (protein molecules) to genetic mutations scientists Beadle & Tatum came up with the “one gene-one enzyme” gene theory. Each protein is specific to that gene and to that organism.

Beadle and Tatum set out to provide experimental proof of the connection between genes and enzymes. They hypothesized that if there really was a one-to-one relationship between genes and specific enzymes, it should be possible to create genetic mutants that are unable to carry out specific enzymatic reactions. To test this theory, they exposed spores of Neurospora crassa (a bread mold) to X-rays or UV radiation and studied the resulting mutations. The mutant molds had a variety of special nutritional needs. Unlike their normal counterparts, they could not live without the addition of particular vitamins or amino acids to their food. For example, normal Neurospora requires only one vitamin (biotin), but mutants were created that also required thiamine or choline.

Genetic analysis showed that each mutant differed from the original, normal type by only one gene. Biochemical studies showed that the mutants seemed to be blocked at certain steps in the normal metabolic pathways. Their cells contained large accumulations of the substance synthesized just prior to the blockage point

As Beadle and Tatum had predicted, they were able to create single gene mutations that incapacitated specific enzymes, so that the molds with these mutations required an external supply of the substance that the enzyme normally produced, and the substance that the enzyme normally used, piled up in the cell. These results led them to the one gene/one enzyme hypothesis, which states that each gene is responsible for directing the building of a single, specific enzyme.

B. Manufacture of Proteins

Nucleus of cell

Chromosomes – parts of the cell which carry hereditary information.

DNA – DNA molecules make up chromosomes. The DNA molecules transfer the information that determines the composition of proteins.

Gene – portion of the DNA molecule which contains the set of instructions for manufacturing a specific protein.

Codons – long series of codons makes up a gene.

3 nucleotides – adenine, thymine, cytosine, and guanine.

Amino acid – 3 nucleotides (codon) on the mRNA specify for a specific amino acid transported to ribosomes by tRNA (note: some amino acids can be specified by more than one codon).

Polypeptide – amino acids linked together via peptide bonds to form a polypeptide called a protein.

Protein

C. RNA – ribonucleic acid

Like DNA, RNA is a polynucleotide chain containing only four different types of nucleotides. However, there are four major differences between these two molecules:

1. RNA is a single stranded molecule.

2. DNA has a ribose sugar that lacks one oxygen atom (the meaning of the name “deoxyribose”), compared to RNA’s “ribose” which has a full complement of oxygen.

3. In RNA the nitrogen base uracil (U) – (a single ring pyrimidines) replaces the thymine found in DNA.

• What is the RNA strand corresponding to the DNA strand below?

• ACC GTC CCT CAT AGT CGT AAT CGT ACC GTT ACC GAC

•

4. RNA may be found in the nucleus or the cytoplasm, but DNA never leaves the nucleus.

D. Types of RNA

There are three types of RNA

1. mRNA – messenger RNA – copies a portion of the DNA and takes it to the ribosome where the message will be read.

2. tRNA – transfer RNA – picks up amino acids and takes them to the ribosome to create the polypeptide. Figure to the right.

3. rRNA – ribosomal RNA - located in the ribosome. The function of the rRNA is to provide a mechanism for decoding mRNA into amino acids and to interact with the tRNA’s during translation.

E. Protein Synthesis

Protein synthesis involves two processes:

1. Transcription

2. Translation

There can be thousands of amino acids in a single protein chain. The sequence of amino acids determines the type of protein. It is a very sensitive process, if even one amino acid is out of sequence it can affect the entire protein. For example, sickle cell anemia occurs because the amino acid valine is substituted for the amino acid glutamic acid.

1. Transcription

Since DNA cannot leave the nucleus, a copy of a gene must be made. One strand of the DNA is copied onto RNA.

Transcription can be divided into three distinct phases.

a. Initiation

• RNA polymerase binds to the segment to be transcribed and opens the double helix.

• RNA binds to a region in front of a gene termed the promoter. This indicates which strand to transcribe and where RNA polymerase should start.

b. Elongation

• The formation of mRNA by RNA polymerase

• This is similar to DNA replication, except the mRNA contains uracil instead of thymine.

c. Termination

• RNA polymerase stops transcribing a gene when it reaches the termination sequence of bases.

• Transcription stops and the mRNA strand peels off the DNA and moves to a ribosome in the cytoplasm.

• After the copy is released, the DNA zips back up.

[pic]

Characteristics of the Genetic Code

mRNA Codon Table

|First base |Second base |Third base |

| |U |

|helicase |cleaves and unwinds short sections of DNA ahead of the replication fork |

|primase |synthesizes an RNA primer to begin the elongation process |

|DNA polymerase |adds new nucleotides to the 3’ OH group of an existing nucleotide strand; dismantles the RNA primer; proofreads base |

| |pairing |

|DNA ligase |splices together Okazaki fragments in the lagging strand |

|Amino acid |Three-letter abbreviation |Amino acid |Three-letter abbreviation |

|alanine |ala |leucine |leu |

|arginine |arg |lysine |lys |

|asparagine |asn |methionine |met |

|aspartate |asp |phenylalanine |phe |

|cysteine |cys |proline |pro |

|glutamate |glu |serine |ser |

|glutamine |gln |threonine |thr |

|glycine |gly |tryptophan |trp |

|histidine |his |tyrosine |tyr |

|isoleucine |ile |valine |val |

Amino acid abbreviations

Procedure

1. The illustration shows an imaginary polypeptide produced by a bacterial cell. Using the tables above, draw one possible nucleotide sequence for the DNA molecule that contains the gene for this polypeptide.

2. Translation

• This is the actual reading of the code and the construction of the protein. For a cell to synthesize a particular polypeptide, it must assemble the amino acid building blocks in a specific sequence. This sequence determines the molecules overall structure and shape, and therefore, its characteristics.

• The sequence of codons on the mRNA directs the assembly of the polypeptide, and ensures that the component amino acids are brought together in the proper sequence. The message carried by the mRNA is rewritten in the sequence of amino acids found in the newly constructed polypeptide.

• This process is called translation, and consists of three distinct stages.

a) Initiation

• The ribosome binds to the mRNA at the start codon.

• The start (initiator) codon AUG codes for methionine, meaning that all polypeptides begin with the amino acid methionine (Met).

b) Elongation

• A molecule of transfer RNA (tRNA) serves as an amino acid carrier. Each tRNA molecule has a site where it can bond to one specific amino acid (A site).

• It also has a triplet sequence of nucleotides, called an anticodon, which is complimentary to one particular codon found on the mRNA. This site on the mRNA is called the P site.

• As the tRNA molecule carries an amino acid to the mRNA on the ribosome, it will only deliver its cargo to one specific address (P site). This address is the codon, which matches the complimentary sequence of bases on the anticodon.

• A peptide bond will form between the two amino acids.

• The ribosome will move down one codon, so that the second codon on the mRNA can be read. The corresponding tRNA will be sent out to carry the appropriate amino acid back to the ribosome.

[pic]

c) Termination

The ribosome reaches a stop codon (UAA, UAG, UGA).

These codons do not code for an amino acid, which causes the protein synthesis to stop, releasing the finished protein.

The mRNA may be read hundreds of times forming many copies of the same protein.

[pic]

Summary

1. Messenger RNA is formed in the nucleus (transcription). It is a sequence of nucleotides which is complementary to a section of the DNA strand.

2. The mRNA leaves the nucleus and becomes associated with a ribosome. The ribosome is the site of polypeptide synthesis. The ribosome consists of ribosomal RNA (rRNA), and many different proteins. The rRNA serves as a framework for the ribosomal proteins. Together, these molecules play an important role in binding the mRNA and tRNA molecules.

3. In the cytoplasm there is a pool of amino acids. Each kind of amino acid is linked to one specific enzyme for each amino acid. ATP supplies the energy.

4. Each tRNA molecule carries its amino acid to the ribosome. The anticodon of the tree specific nucleotides on the tRNA determines where on the mRNA it will fit. If the mRNA codon reads AAG, what must the anticodon read?

5. As the amino acids are brought together on the ribosome in sequence, a peptide bond forms between adjacent amino acid molecules.

6. The tRNA molecule is released into the cytoplasm. It is now free to pick up another of the same kind of amino acid.

7. When the mRNA codon reads “stop” (UAG, UAA, or UGA), the polypeptide is released. The mRNA is also released from the ribosome.

A) DNA codons ATG TCA CCG AAG CGT

TAC AGT GGC TTC GCA

TRANSCRIPTION

B) mRNA codons AUG UCA CCG AAG CGU

TRANSLATION

C) Protein MET SER PRO LYS ARG

A short segment of DNA is shown in (A). Each DNA codon consists of three nucleotide pairs. For transcription, one strand of the DNA will serve as a template to make messenger RNA (mRNA) – (b). The segment of mRNA corresponds to the DNA segment above. (C) – The segment of protein corresponds to the codons in the mRNA, and consequently, to the information in the DNA. Each specific amino acid is indicated by its short form, eg., LYS = lystine

[pic]

[pic]

Simulating Protein Synthesis

Experimental Plan

1. As a group, list the steps that are involved in transcription and translation. For each step, note the structures, molecules, and events involved.

2. Discuss how you might simulate transcription and translation in your classroom. Your simulation could take any form. For example, you could prepare an interactive computer program, write and perform a play, a story or construct a physical model.

3. Once you have agreed on a plan, list the materials and equipment you will need to carry out your simulation. Assign responsibilities to each member of your group. Then assemble your materials and prepare your simulation.

Data and Observations

4. Present your simulation to the class. Record any comments you receive from your classmates.

Transcription and Translation Worksheet

Answer the following questions in the space provided.

1. Name the amino acids that correspond with the following mRNA codons:

a) AGA ________________________

b) GCC_________________________

c) CUU ________________________

d) UGA ________________________

2. A geneticist isolates a strand of DNA containing the following nucleotide sequence:

TACGGTCACATGATT

a) Provide the nucleotide sequence of the mRNA strand transcribed from this sequence.

_____________________________________________________________________

b) What is the amino acid sequence of the polypeptide produced from this strand of mRNA?

_____________________________________________________________________

c) What is the nucleotide sequence of the tRNA anticodon that codes for the first amino acid in the polypeptide?

____________________________________________________________________

3. The same geneticist then isolates the following polypeptide: met-lys-his-trp.

a) What amino acids make up this polypeptide?

_____________________________________________________________________

b) How many different nucleotide sequences could code for this polypeptide? List these sequences.

_____________________________________________________________________

_____________________________________________________________________

_____________________________________________________________________

_____________________________________________________________________

_____________________________________________________________________

c) Which important characteristic of the genetic code is illustrated in b)? How does this characteristic reduce the number of amino acids that are incorrectly translated?

_____________________________________________________________________

_____________________________________________________________________

_____________________________________________________________________

_____________________________________________________________________

_____________________________________________________________________

F. Sorting and Analyzing DNA

Gel electrophoresis is used to separate molecules according to their mass and charge. It is used to separate fragments produced by using restriction enzymes. A solution that contains DNA fragments is applied to one end of the gel. An electric current is then passed through the gel. One end of the gel will develop a positive electric charge, the other a negative electric charge.

DNA will be attracted to the positive charge as it is negatively charged. Smaller fragments move more frequently. Fragments will separate into a pattern of bands, a DNA fingerprint. Different samples that display similar DNA fragments provided evidence of inheritance. This is used in crime scene investigation and paternity cases.

[pic]

G. DNA Fingerprinting

A DNA fingerprint is a pattern of DNA fragments that results when DNA undergoes a process known as gel electrophoresis. A solution containing DNA fragments is applied at one end of a gel, and an electric current is passed through it, causing a positive electric charge to develop at one end of the gel and a negative electric charge to form at the other. DNA has a negative charge, and the fragments thus move toward the gel’s positive end. Smaller fragments move more quickly than larger ones. As a result, the fragments separate into a pattern of bands, a DNA fingerprint.

Once a DNA fingerprint has been created, scientists identify fragments of DNA that contain sequences that are unique to an individual. These sequences are known as VNTRs, or variable number tandem repeats. They contain 20-100 base pairs and are found in the non-coding regions of human DNA. VNTRs are inherited from parents. This means that some of your VNTRs came from your mother and some from your father. Your pattern of VNTRs will be unique to you, however.

As a result of this unique pattern, DNA fingerprinting can be used to identify an individual. How is this done? While DNA fingerprinting cannot distinguish an individual directly, it can do so through means of comparison. For example, a DNA fingerprint can help determine if two DNA samples are from the same person. This is useful in solving crimes in which DNA evidence, such as hair, blood, or skin tissue, has been found at the scene. If the DNA fingerprint of a suspect matches the DNA fingerprint found at the crime scene, this indicates that the individual was likely to have been at the scene of the crime. It’s important to note that DNA fingerprinting is not foolproof. It can only determine that there is an extremely high probability that the DNA run though the gel belongs to a certain person. Technical errors can occur in the lab. The margin for error increases if the DNA sample is very small. Also, until recently, strict lab standards for DNA testing were not universal.

DNA fingerprinting can also help determine the paternity or maternity of a child, as you will learn in Thought Lab 18.5: Reading a DNA Fingerprint. As indicated, a person’s VNTRs are inherited from both parents. By comparing a child’s DNA fingerprint with those of the adults, the child’s parent(s) can be identified. The following diagram shows the results of a gel electrophoresis analysis of one child and four different sets of parents. Use these DNA fingerprints to complete Thought Lab 18.5.

[pic]

The following diagram shows the results of a gel electrophoresis analysis of one child and four different sets of parents. Use these DNA fingerprints to answer the Analysis questions and identify the child’s biological parents.

[pic]

Analysis

1. Which parental DNA matches the child’s DNA? How do you know?

2. Try to determine the percentage of the father’s DNA that matches the child’s DNA. Can you do the same for the mother’s DNA? Explain why or why not.

3. Describe other situations in which DNA fingerprinting might be useful.

H. Tracing Ancestry

• The cytoplasm in a zygote is donated by the ovum.

• The sperm contributes essentially no cytoplasm, therefore no cytoplasmic organelles.

• The DNA in the nuclei of your cells is made from equal combinations from your parents, your mitochondrial DNA (mtDNA) is genetically identical to your mothers mtDNA.

• Both mtDNA and chloroplast DNA is independent of the nuclei DNA.

• If two people have identical mtDNA sequences, they likely share a relatively recent maternal ancestor.

• By comparing mtDNA of different living people, scientists can deduce lineage patterns that reveal prehistoric relationships among human populations.

• As well, as evolutionary paths of animals and plant species can be determined.

Detection

Amniocentesis

[pic]

Chorionic Villi Sampling

[pic]

I. Mutations

• If genetic information is to be transmitted accurately from one cell generation to the next, then DNA replication must be accurate. If there is a mistake in the process, the sequence of nitrogen bases in the DNA may be altered. This is a mutation. Since every cell carries a full complement of genetic material, a mutation can occur in any cell in the body. However, only mutations, which occur in reproductive cells can be passed on to the next generation.

• Mutations are rare, random events. The likelihood of a specific gene mutation occurring is one in a million per generation. However, if one considers the total number of individuals in a population, and the fact that each individual has about 35,000 genes, there is a good chance that mutations do occur in each generation.

• A low rate of mutation is desirable since it can introduce new characteristics and traits into a population. Too high a rate is potentially harmful because most detectable mutations are harmful rather than beneficial to the organism.

• Mutations, which alter a single gene, are commonly called point mutations. A much larger alteration, which visibly affects the structure and/or number of chromosomes, is classified as a chromosomal mutation.

Point Mutations

There are two main kinds of point mutations: nitrogen base pair substitution and frameshift mutations.

Base Pair Substitution

• In a base pair substitution, only a single base pair is affected. For example, a single G-C pair might be replaced by an A-T pair. If the change is in the third position of the codon, it might not alter the genetic message at all. However, it may change the code and call for a completely different amino acid. This may change the character of the polypeptide chain, sometimes with serious implications.

• The nitrogen base pair mutation may also change a codon to code for a stop in the translation process. This would in effect tell the cell machinery to stop building a polypeptide before it was complete. As a result, the polypeptide would lack some or all of its ability to function properly since it would lack part of its structure.

Point Mutations are categorized into the following:

a) Silent mutation – a change in base pairs that does not result in a change in an amino acid. i.e., cysteine UGU to UGC

b) Missense mutation – a change in base sequence which results in altering the codon leading to a different amino acid. i.e., sickle cell anemia

c) Nonsense mutation – a change in base sequence that causes a stop codon to replace an amino acid codon. Results in a fragmented polypeptide which is often lethal to the cell.

[pic]

Frameshift Mutation

This mutation occurs when one or more nitrogen base pairs are added or deleted from the DNA strand. As you know, the DNA information is read from a starting point in groups or “frames” of three. There is nothing to identify each specific codon so accuracy is important. The cell machinery simply reads the information in groups of threes. If a base is added or deleted, all the codons from that point are affected. This mutation causes a shift in the frames of three, and the cell machinery will now read a completely different code.

[pic]

[pic]

Chromosomal Mutations

Mutation of large segments of DNA and is seen at the chromosomal level.

a) Translocation – the relocation of groups of base pairs from one part of the genome to another . This often occurs between nonhomologous chromosomes. This is believed to be the cause of some types of leukemia.

b) Inversion – section of the chromosome has reversed its orientation in the chromosome. Hunters Syndrome a rare sex-linked hereditary disorder that varies widely in its severity but is generally characterized by some degree of dwarfism, mental retardation, and deafness, is caused by inversion mutations.

What Causes Mutations?

Mutations may arise from either a spontaneous mutation or exposure to a mutagenic agent.

Spontaneous Mutation – occurs under normal conditions. Often caused by mispairing mistakes in which a “wrong’ nucleotide is added during the process of replication. Malfunction of DNA polymerase I and DNA polymerase II.

Mutagenic Agents – occur under artificial conditions, such as exposure to mutagens (agents that increase the natural rate of mutations). Examples include UV radiation, X-rays, and chemicals such as mustard gas.

Mutations Worksheet

Answer the following questions in the space provided.

1. Fill in the blanks with the appropriate terms.

A permanent change in the genetic material of an organisms is called a a)_______________. Permanent genetic changes that occur in body cells are called b) _________________________, while those that occur in reproductive cells are called c) __________________________. Body cell mutations are a key cause of d) __________________. A substance that increases the rate of mutation is called a e) _____________________. When a substance causes physical changes in the structure of DNA, it is called a f) __________________________________. Mutations can also be caused by g) _________________________, which enter the nucleus of a cell and

h) ____________ mutations by reacting chemically with the DNA.

2. Consider the following nucleotide sequence in a strand of mRNA:

GUU-CAU-UUG-CUC-CCG-AAG

val – his – leu – leu – pro – lys

a) The second uracil base in the first leucine in the polypeptide is substituted with an adenine base, resulting in the replacement of the codon UUG with the codon UAG. What type of mutation results from this substitution? Explain your reasoning.

_____________________________________________________________________

_______________________________________________________________________________________________________________________________________________________________________________________________________________

b) The uracil base in the codon for histidine is substituted with a cytosine base. What type of mutation results from this substitution? Explain.

_______________________________________________________________________________________________________________________________________________________________________________________________________________

_____________________________________________________________________

c) The first cytosine base in the second leucine is deleted. Write the nucleotide and amino acid sequences that occur as a result of this mutation. What type of mutation(s) may result from this deletion?

__________________________________________________________________________________________________________________________________________

__________________________________________________________________________________________________________________________________________

Thought Lab – Investigating Cancer Genes

Procedure

1. Study the graphs. Write a brief summary of the relationships shown in the graph.

2. Record your ideas about the molecular reactions that may be occurring based on what you have learned in this chapter.

3. Conduct research to describe one of the molecular reactions that might contribute to the relationship you see in the graph. You may find the following keywords helpful to guide your research:

A. oncogenes

B. tumour-suppressor genes

C. stability genes

D. p53 gene

J. Prevention of Cancer

Smoking

Smoking and second-hand smoke inhalation are the most preventable causes of cancer in Canada. Cigarette smoke is linked to the development of numerous cancers. (This is discussed in the handout FAQ—Tobacco and Cancer in greater detail.) 80 carcinogens are found in cigarette smoke, along with many other toxic compounds. These carcinogens are chemical mutagens. They cause cancer by entering the nucleus of a cell and inducing mutations by reacting chemically with the DNA. A chemical mutagen may act by inserting itself into the DNA molecule in a manner that causes a nucleotide substitution or a frameshift mutation. Other chemical mutagens have a structure that is similar to the structure of ordinary nucleotides but with different base pairing properties. When these mutagens are incorporated into a DNA strand, they can cause incorrect nucleotides to be inserted during DNA replication. Over time, these mutations can result in cancer.

Diet and Exercise

A healthy diet and regular exercise have a big influence on the risk of developing cancer. Next to stopping smoking, eating a healthy diet, maintaining a healthy weight, and getting enough exercise are the most important lifestyle habits a person can maintain to reduce the incidence of cancer. Overweight individuals have a higher risk of developing certain cancers, including breast, colon, kidney, esophageal, and endometrial (referring to the lining of the uterus) cancers. Additionally, people who carry excess body weight produce more insulin and estrogen, hormones that have been linked to accelerated tumour growth. Increasing physical activity and decreasing portion size are two good ways to maintain a healthy weight. The food choices an individual makes can also reduce his or her cancer risk. With regards to cancer prevention, studies have shown that eating at least 5 servings of fruits and vegetables a day reduces the risk of many cancers, including cancers of the lung and digestive system. Research has shown that brightly coloured fruits and vegetables have a particularly high level of cancer-fighting nutrients. Consumption of red and processed meats is linked to an increased risk of cancer.

Sun Exposure

Ultraviolet (UV) radiation is a known physical mutagen. Physical mutagens cause physical changes

in the structure of DNA. They tear through DNA molecules, causing random changes that range from point mutations to the loss of large portions of chromosomes. The resulting mutations can lead to tumour formation. Increased risk of skin cancers, such as the highly curable basal and squamous cell (types of skin cells) cancers, as well as the more dangerous melanoma, is indisputably linked to exposure to UV radiation, both from the sun and artificial tanning. The best way to reduce this risk is to limit sun exposure to short periods of time when the sun is less intense and to “practice safe sun” by wearing sunscreen and protective clothing. X-ray radiation is an even more powerful physical mutagen than UV radiation, and exposure to these rays should be limited if possible.

Environment

Today’s society is home to many carcinogens. These include chemical mutagens, such as many chemical additives, environmental pollutants, drugs, and hormonal treatments; physical mutagens, including most forms of radiation; and even infectious diseases, such as bacteria, viruses, and parasites. Infectious disease may increase cancer risk by compromising the immune system, causing long-term inflammation in the body, or by directly interfering with the body’s DNA. While some infectious diseases have been found to play a role in the development of cancer, it is important to bear in mind that most people who get these infections do not go on to develop cancer. Certain varieties of HPV (human papilloma virus) have been linked to cervical cancer, the second most common cancer in women, as well as cancer of the penis, vagina, and anus, among others. A vaccine that prevents four of the most common forms of HPV (two-high risk types and two low-risk types) has been available in Canada since 2006. While the vaccine has been shown to provide protection against these four forms of the virus, it will not treat a pre-existing infection.

Early Detection

A last, but important, means of preventing cancer is early detection. In some cancers, such as cervical cancer, pre-cancerous cells can be easily detected. Prompt treatment in cases of early detection can often stop tumour formation. Even in cases where cancer has already developed, early detection can significantly increase the cure rate or future life expectancy of an individual.

K. Restriction Endonucleases

[pic]

L. Cohen-Boyer Experiment

1973 was the year in which genetic engineering was born. In this year, the American researchers Stanley Cohen and Herbert Boyer created the first genetically engineered organism. This organism, known as a chimera, consisted of a bacterium that contained DNA from an unrelated species, Xenopus laevis, the African clawed toad. To create this chimera, Cohen and Boyer first isolated a bacteria plasmid known as pSC101 (so named as it was the 101st plasmid isolated by Stanley Cohen). A plasmid is a double stranded, circular molecule of DNA that replicates on its own, independently of the bacterial chromosomal DNA. Plasmid pSC101 contains a derivative of an R factor, a factor that codes for antibiotic resistance in bacteria, in this case to tetracycline. In general, R factors are very large and can be cleaved at many sites by restriction enzymes. Plasmid pSC101 contains a derivative of an R factor, which has only 9000 base pairs, and is cleaved in only one site by the restriction endonuclease EcoRI. When cleaved, the plasmid opens at this specific site to form a linear piece of double stranded DNA with sticky ends on both ends. Another gene that is cut by restriction endonuclease EcoRI may then bind to these sticky ends and be added to the plasmid.

Over 30 years ago, Cohen and Boyer created their chimera in just this way. The scientists isolated a gene from Xenopus laevis that coded for the production of rRNA. This gene was cleaved with restriction endonuclease EcoRI and inserted into plasmid pSC101. The plasmid now contained the amphibian gene for rRNA production and the bacterial gene for tetracycline resistance. Bacteria were then exposed to both the recombinant plasmid and to tetracycline. Those bacteria that displayed tetracycline resistance had taken up the plasmid.

The following figure illustrates the Cohen-Boyer experiment. In the figure, the amphibian gene coding for the production of rRNA is shown in black and the bacterial gene, tetR, which confers resistance to the antibiotic tetracycline, is shown in white. The restriction endonuclease EcoR1 and DNA ligase were used to splice (insert) a gene from the toad into the plasmid pSC101.

Thought Lab – Recreating the First Chimera

In genetic engineering, a chimera is a genetically engineered organism that contains DNA from unrelated species. The first chimera was created in 1973 by the American team of Stanley Cohen and Herbert Boyer. Bacteria were then exposed to the recombinant plasmid. Those bacteria that displayed tetracycline resistance had taken up the plasmid.

In Cohen and Boyer’s experiment, the amphibian gene coded for the production of rRNA. The bacterial gene tetR conferred resistance to the antibiotic tetracycline. They used the restriction endonuclease EcoR1 and DNA ligase to splice (insert) a gene from a toad into a molecule of bacterial DNA plasmid pSC101.

Procedure

1. Study the illustration of the Cohen-Boyer experiment. Make a list of the materials that the researchers used.

2. Develop a plan to simulate the experiment. Show how you will use materials in your classroom to represent the materials that Cohen and Boyer used. Then perform your simulation.

Analysis

1. How did your simulation illustrate the action of an endonuclease and a ligase? In what ways was your simulation effective? What were its limitations?

2. The Cohen-Boyer experiment was important because it created a colony of bacterial cells that were resistant to the antibiotic tetracycline and produced amphibian rRNA. What other bacterial phenotypes would have resulted from this experiment? What would each phenotype indicate about events at the molecular level?

3. a) Give one example of how you might use this technology for a social or industrial purpose.

b) What environmental, social, or ethical issues would your experiment raise? Make a list of these issues, and discuss them with other students in your class.

................
................

In order to avoid copyright disputes, this page is only a partial summary.

Google Online Preview   Download