Curriculum Map 146 - BIOLOGY JUNCTION

Cell Respiration

Cell Respiration

Overview:
In this experiment, you will work with seeds that are living but dormant. A seed contains an embryo plant and a food supply surrounded by a seed coat. When the necessary conditions are met, germination occurs, and the rate of cellular respiration greatly increases. In this experiment you will measure oxygen consumption during germination. You will measure the change in gas volume in respirometers containing either germinating or non-germinating pea seeds. In addition, you will measure the rate of respiration of these peas at two different temperatures.

Objectives:
Before doing this laboratory you should understand:

how a respirometer works in terms of the gas laws; and
the general processes of metabolism in living organisms.

After doing this laboratory you should be able to:

calculate the rate of cell respiration from experimental data.
relate gas production to respiration rate; and
test the effect of temperature on the rate of cell respiration in ungerminated versus germinated seeds in a controlled experiment.

Introduction:
Cellular respiration is the release of energy from organic compounds by metabolic chemical oxidation in the mitochondria within each cell. Cellular respiration involves a series of enzyme-mediated reactions. The equation below shows the complete oxidation of glucose. Oxygen is required for this energy-releasing process to occur.

C6H12O6 + 6O2 —–> 6 CO2 + 6 H2O + 686 kilocalories of energy / mole of glucose oxidized

By studying the equation above, you will notice there are three ways cellular respiration could be measured. One could measure the:

1. Consumption of O2 ( How many moles of oxygen are consumed in cellular respiration?)

2. Production of CO2 ( How many moles of carbon dioxide are produced by cellular respiration?)

3. Release of energy during cellular respiration.

In this experiment, the relative volume of O2 consumed by germinating and non-germinating (dry) peas at two different temperatures will be measured.

Background Information:
A number of physical laws relating to gases are important to the understanding of how the apparatus that you will use in this exercise works. The laws are summarized in the general gas law that states:

PV = nRT

where

P is the pressure of the gas,

V is the volume of the gas,

n is the number of molecules of gas,

R is the gas constant ( its value is fixed), and

T is the temperature of the gas (in K0).

This law implies the following important concepts about gases:

1. If temperature and pressure are kept constant, then the volume of the gas is directly proportional to the number of molecules of gas.

2. If the temperature and volume remain constant, then the pressure of the gas changes in direct proportion to the number of molecules of gas present.

3. If the number of gas molecules and the temperature remain constant, then the pressure is inversely proportional to the volume.

4. If the temperature changes and the number of gas molecules is kept constant, then either pressure or volume ( or both ) will change in direct proportion to the temperature.

It is also important to remember that gases and fluids flow from regions of high pressure to regions of low pressure.

In this experiment, the CO2 produced during cellular respiration will be removed by potassium hydroxide (KOH) and will form solid potassium carbonate (K2CO3) according to the following reaction.

CO2 + 2 KOH —-> K2CO3 + H2O

Since the carbon dioxide is being removed, the change in the volume of gas in the respirometer will be directly related to the amount of oxygen consumed. In the experimental apparatus if water temperature and volume remain constant, the water will move toward the region of lower pressure. During respiration, oxygen will be consumed. Its volume will be reduced, because the carbon dioxide produced is being converted to a solid. The net result is a decrease in gas volume within the tube, and a related decrease in pressure in the tube. The vial with glass beads alone will permit detection of any changes in volume due to atmospheric pressure changes or temperature changes. The amount of oxygen consumed will be measured over a period of time. Six respirometers should be set up as follows:

Respirometer	Temperature	Contents
1	Room	Germinating seeds
2	Room	Dry Seeds and Beads
3	Room	Beads
4	100C	Germinating Seeds
5	100C	Dry Seeds and Beans
6	100C	Beads

Procedure:
1.Prepare a room-temperature bath (approx. 25 degrees Celsius) and a cold-water bath (approx. 10 degrees Celsius).

2.Find the volume of 25 germinating peas by filling a 100mL graduated cylinder 50mL and measuring the displaced water.

3.Fill the graduated cylinder with 50mL water again and drop 25 non-germinating peas and add enough glass beads to attain an equal volume to the germinating peas.

4.Using the same procedure as in the previous two steps, find out how many glass beads are required to attain the same volume as the 25 germinating peas.

5.Repeat steps 2-4. These will go into the 10-degree bath.

6.To assemble 6 respirometers, obtain 6 vials, each with an attached stopper and pipette. Number the vials. Place a small wad of absorbent cotton in the bottom of each vial and, using a dropper, saturate the cotton with 15% KOH (potassium hydroxide). It is important that the same amount of KOH be used for each respirometer.

7.Place a small wad of dry, nonabsorbent cotton on top of the saturated cotton.

8.Place the first set of germinating peas, dry peas & beads, and glass beads in the first three vials, respectively. Place the next set of germinating peas, dry peas & beads, and glass beads in vials 4, 4, and 6, respectively. Insert the stopper with the calibrated pipette. Seal the set-up with silicone or petroleum jelly. Place a weighted collar on each end of the vial. Several washers around the pipette make good weights.

9.Make a sling of masking tape attached to each side of the water baths. This will hold the ends of the pipettes out of the water during an equilibration period of 7 minutes. Vials 1, 2, and 3 should be in the room temperature bath, and the other three should be in the 10 degree bath.

10.After 7 min., put all six set-ups entirely into the water. A little water should enter the pipettes and then stop. If the water continues to enter the pipette, check for leaks in the respirometer.

11.Allow the respirometers to equilibrate for 3 more minutes and then record the initial position of the water in each pipette to the nearest 0.01mL (time 0). Check the temperature in both baths and record. Record the water level in the six pipettes every 5 minutes for 20 minutes.

Table 5.1: Measurement of O2 Consumption by Soaked and Dry Pea Seeds at Room Temperature (250C) and 100C Using Volumetric Methods.

Temp (oC)	Time (min)	Beads Alone		Germinating Peas		Dry Peas and Beans
		Reading at time X	Diff*	Reading at time X	Diff*	Corrected Diff. ^	Reading at time X	Diff*	Corrected diff ^
	Initial – 0
	0-5
	5- 10
	10 -15
	15-20
	Initial – 0
	0-5
	5- 10
	10 -15
	15-20

* difference = ( initial reading at time 0) – ( reading at time X )

^ corrected difference = ( initial pea seed reading at time 0 – pea seed reading at time X) – ( initial bead reading at time X).

Analysis of Results:
1. In this investigation, you are investigating both the effect of germination versus non-germination and warm temperature versus cold temperature on respiration rate. Identify the hypothesis being tested in this activity.

_______________________________________________________________________

2. This activity uses a number of controls. Identify at least three of the control, and describe the purpose of each control.

_______________________________________________________________________

3. Graph the results from the corrected difference column for the germinating peas and dry peas at both room temperature and 100C.

a. What is the independent variable? ____________________________________________________

b. What is the dependent variable? ______________________________________________________

Graph Title: _____________________________________________________________________

Graph 5.1

4. Describe and explain the relationship between the amount of oxygen consumed and time.

_______________________________________________________________________

5. From the slope of the four lines on the graph, determine the rate of oxygen consumption of germinating and dry peas during the experiments at room temperature and 100C. Recall that rate = delta Y/delta X.

Table 5.2

Condition	Show Calculations Here	Rate in ml.O2 / min
Germinating Peas/100C
Germinating peas /Room Temperature
Dry peas/100C
Dry Peas /Room Temperature

6. Why is it necessary to correct the readings from the peas with the readings from the beads?

_______________________________________________________________________

7. Explain the effect of germination ( versus non-germination) on peas seed respiration.

_______________________________________________________________________

8. What is the purpose of KOH in this experiment?

_______________________________________________________________________

9. Why did the vial have to be completely sealed around the stopper?

_______________________________________________________________________

10. If you used the same experimental design to compare the rates of respiration of a 25 g. reptile and a 25 g. mammal, at 100C, what results would you expect/ Explain your reasoning.

_______________________________________________________________________

11. If respiration in a small mammal were studied at both room temperature (210C) and 100C, what results would you predict? Explain your reasoning.

_______________________________________________________________________

12. Explain why water moved into the respirometer pipettes.

_______________________________________________________________________

________________________________________________________________________

AP LAB PAGE

DNA & Protein Synthesis Chapter 10 Worksheet

DNA & Protein Synthesis

Section 10-1 DNA

1. What does DNA stand for?

2. What is DNA’s primary function?

3. What is the function of proteins?

4. What are the repeating subunits called that make up DNA?

5. Name the 3 parts of a DNA nucleotide.

6. Sketch and label a DNA nucleotide.

7. Name the 4 nitrogen bases on DNA.

8. What is the difference between a purine & a pyrimidine?

9. Name 2 purines.

10. Name 2 pyrimidines.

11.Who is responsible for determining the structure of the DNA molecule & in what year was this done?

12. The model of DNA is known as a ____________________________ because it is composed of two ___________________ chains wrapped around each other.

13. What makes up the sides of a DNA molecule?

14. What makes up the “steps” of a DNA molecule?

15. How did Rosalind Franklin contribute to determining the structure of DNA?

16. What type of bonds holds the DNA bases together? Are they strong or weak bonds?

17. What makes up the “backbone” of the DNA molecule?

18. On DNA, a ____________________ base will always pair with a __________________ base.

19. What is the most common form of DNA found in organisms?

20. How many base pairs are in a full turn or twist of a DNA molecule?

21. Name the complementary base pairs on DNA.

22. How many hydrogen bonds link cytosine & guanine? adenine & thymine?

23. How does the nucleotide sequence in one chain of DNA compare with the other chain of DNA?

24. Why must DNA be able to make copies of itself?

25. Define DNA replication.

26. What is the first step that must occur in DNA replication?

27. What acts as the template in DNA replication?

28. What is a replication fork?

29. What enzymes help separate the 2 strands of nucleotides on DNA? What bonds do they break?

30. What is the function of DNA polymerases?

31. ____________________ are joined to replicating strands of DNA by ________________ bonds.

32. If the sequence of nucleotides on the original DNA strand was A – G – G – C – T – A, what would be the nucleotide sequence on the complementary strand of DNA?

33. Does replication of DNA begin at one end and proceed to the other? Explain.

34. Why does DNA replication take place at many places on the molecule simultaneously?

35. When replication is complete, how do the 2 new DNA molecules compare to each other & the original DNA molecule?

36. Is DNA replicated (copied) before or after cell division?

37. Sketch & label DNA replication. (Figure 10-5, page 188)

38. What is the error rate in DNA replication? What helps lower this error rate to 1 in 1 billion nucleotides?

39. What is a mutation?

40. Name several things that can cause DNA mutations.

Section 10-2 RNA

41. What sugar is found on DNA?

42. What base is missing on RNA, & what other base replaces it?

43. Uracil will pair with what other on DNA?

44. Is RNA double or single stranded?

45. Name the 3 types of RNA and tell the shape of each.

46. Which type of RNA copies DNA’s instructions in the nucleus?

47. Which type of RNA is most abundant?

48. What does tRNA transport?

49. What 2 things make up ribosomes?

50. Define transcription.

51. In what part of a cell are proteins made?

52. What is RNA polymerase & tell its function.

53. What are promoters?

54. Where does RNA polymerase bind to the DNA it is transcribing?

55.What makes the beginning of a new gene on DNA in eukaryotes?

56. What do promoters mark the beginning of on prokaryotic DNA?

57. When a promoter binds to DNA, What happens to the double helix?

58. Are both strands of DNA copied during transcription?

59. As RNA polymerase moves along the DNA template strand, what is being added?

60. What bases pair with each other during transcription?

61. What is the termination signal?

62. What happens when RNA polymerase reaches the termination signal?

63. What are the products of transcription called?

64. Transcripts are actually ____________________________ molecules.

65. In transcription, ________________________’s instructions for making a protein

are copied by _______________________.

66. Which RNA molecules are involved in the synthesis (making) of a protein?

67. What happens to the newly made mRNA molecule following transcription in the nucleus?

Section 10-3 Protein Synthesis

68. What makes up proteins, what are the subunits called, & what bonds them together?

69. How many different kinds of amino acids make up proteins?

70. What determines how protein polypeptides fold into 3-dimensional structures?

71. Why does a protein need a 3-dimensional structure?

72. What is the genetic code & why is it important?

73. What is a codon & what does each codon code for?

74. How many codons exist?

75. Name the amino acid coded for by each of these codons:

a. UUA

b. AUU

c. UGU

d. AAA

e. GAG

f. UAA

76. What codon starts protein synthesis?

77. What codons stop protein synthesis?

78. Proteins are synthesized (made) at what organelle in the cytosol?

79. Sketch and label a tRNA molecule & tell its function.

80. Define translation & tell how it starts.

81. Where are amino acids found in a cell & how are they transported?

82. What is an anticodon & where is it found on tRNA?

83. What codon on mRNA would bind with these anticodons: (use table 10-1, page 194)

a. AAA

b. GGA

c. UAC

d. CGU

84. What are ribosomes made of and in what 2 places can they be found in a cell?

85. What is the difference between proteins made by free ribosomes & those made by attached, membrane proteins on the ER?

86. How many binding sites are found on the ribosomes and what does each site hold?

87. To start making a protein or _________________________________, a ribosome attaches to the ______________________________ codon on the __________________ transcript.

88. The start codon, AUG, pairs with what anticodon on a tRNA molecule?

89. What amino acid does the start codon always carry?

90. What type of bonds are the ones that attach amino acids to each other in a growing polypeptide?

91. __________________________ are linked to make proteins as a ______________________ moves along the mRNA transcript.

92. What ends translation?

93. Can more than one ribosome at a time translate an mRNA transcript? Explain.

94. What determines the primary structure of a protein?

95. What would the translation of these mRNA transcripts produce?

a. UAA CAA GGA GCA UCC

b. UGA CCC GAU UUC AGC

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Floating Leaf Disk Assay

The Floating Leaf Disk Assay for Investigating Photosynthesis

Brad Williamson

Introduction:

Trying to find a good, quantitative procedure that students can use for exploring photosynthesis is a challenge. The standard procedures such as counting oxygen bubbles generated by an elodea stem tend to not be “student” proof or reliable. This is a particular problem if your laboratory instruction emphasizes student-generated questions. Over the years, I’ve found that the floating leaf disk assay technique to be reliable and understandable to students. Once the students are familiar with the technique they can readily design experiments to answer their own questions about photosynthesis. I plan to add to this page as I have time to elaborate on the technique and provide suggestions for modifications.

Materials:

1. Sodium bicarbonate (Baking soda)

2. Liquid Soap

3. Plastic syringe (10 cc or larger)—remove any needle!

4. Leaf material

5. Hole punch

6. Plastic cups

7. Timer

8. Light source

Optional:

Buffer Solutions

Colored Cellophane or filters

Leaf material of different ages

Variegated leaf material

Clear Nail polish

Procedure:

Prepare 300 ml of bicarbonate solution for each trial.
1. The bicarbonate serves as an alternate dissolved source of carbon dioxide for photosynthesis. Prepare a 0.2% solution. (This is not very much—it’s about 1/8 of a teaspoon of baking soda in 300 ml of water.) Too much bicarbonate will cause small bubbles (CO2)to form on the surface of the leaf which will make it difficult to sink the leaf disk.
2. Add 1 drop of dilute liquid soap to this solution. The soap wets the hydrophobic surface of the leaf allowing the solution to be drawn into the leaf. It’s difficult to quantify this since liquid soaps vary in concentration. Avoid suds. If your solution generates suds then dilute it with more bicarbonate solution.

Cut 10 or more uniform leaf disks for each trial

1. Single hole punches work well for this but stout plastic straws will work as well
2. Choice of the leaf material is perhaps the most critical aspect of this procedure. The leaf surface should be smooth and not too thick. Avoid plants with hairy leaves. Ivy, fresh spinach, Wisconsin Fast Plant cotyledons—all work well. Ivy seems to provide very consistent results. Any number of plants work. My classes have found that in the spring, Pokeweed may be the best choice.
3. Avoid major veins.

Infiltrate the leaf disks with sodium bicarbonate solution.

1. Remove the piston or plunger and place the leaf disks into the syringe barrel. Replace the plunger being careful not to crush the leaf disks. Push on the plunger until only a small volume of air and leaf disk remain in the barrel (< 10%).

1. Pull a small volume of sodium bicarbonate solution into the syringe. Tap the syringe to suspend the leaf disks in the solution.

1. Holding a finger over the syringe-opening, draw back on the plunger to create a vacuum. Hold this vacuum for about 10 seconds. While holding the vacuum, swirl the leaf disks to suspend them in the solution. Let off the vacuum. The bicarbonate solution will infiltrate the air spaces in the leaf causing the disks to sink. You will probably have to repeat this procedure several times in order to get the disks to sink. You may have difficulty getting the disks to sink even after applying a vacuum three or four times. Generally, this is usually an indication that you need more soap in the bicarbonate solution. Some leaf surfaces are more water repellent than others are. Adding a bit more soap usually solves the problem.

Pour the disks and solution into a clear plastic cup. Add bicarbonate solution to a depth of about 3 centimeters. Use the same depth for each trial. Shallower depths work just as well.

1. This experimental setup includes a control. The leaf disks in the cup on the right were infiltrated with a water solution with a drop of soap—no bicarbonate.

Place under the light source and start the timer. At the end of each minute, record the number of floating disks. Then swirl the disks to dislodge any that are stuck against the sides of the cups. Continue until all of the disks are floating.

1. The control is on the left in each image. In the experimental treatment, on the right, leaf disks are rising and floating on the surface.

Sample results:

Time (minutes)	Disk Floating
1	0
2	0
3	0
4	0
5	0
6	0
7	1
8	1
9	1
10	1
11	4
12	7
13	8
14	10

The point at which 50% of the leaf disks are floating is the point of reference for this procedure. By interpolating from the graph, the 50% floating point is about 11.5 minutes. Using the 50% point provides a greater degree of reliability and repeatability for this procedure.

Genetic Problems Solutions Campbell Ch14

Genetics Problems Campbell 1. A man with hemophilia (a recessive , sex-linked condition has a daughter of normal phenotype. She marries a man who is normal for the trait. What is the probability that a daughter of this mating will be a hemophiliac? A son? If the couple has four sons, what is the probability that all four will be born with hemophilia?

Solution

2. Pseudohypertropic muscular dystrophy is a disorder that causes gradual deterioration of the muscles. It is seen only in boys born to apparently normal parents and usually results in death in the early teens. (a) Is pseudohypertrophic muscular dystrophy caused by a dominant or recessive allele? (b) Is its inheritance sex-linked or autosomal? (c) How do you know? Explain why this disorder is always seen in boys and never girls.

Solution

3. Red-green color blindness is caused by a sex-linked recessive allele. A color-blind man marries a woman with normal vision whose father was color-blind. (a) What is the probability that they will have a color-blind daughter? (b) What is the probability that their first son will be color-blind? (Note: the two questions are worded a bit differently.)

Solution

4. A wild-type fruit fly (heterozygous for gray body color and normal wings was mated with a black fly with vestigial wings. The offspring had the following phenotypic distribution: wild type, 778; black-vestigial, 785; black-normal, 158; gray-vestigial, 162. What is the recombination frequency between these genes for body color and wing type.

Solution

5. In another cross, a wild-type fruit fly (heterozygous for gray body color and red eyes) was mated with a black fruit fly with purple eyes. The offspring were as follows: wild-type, 721; black-purple, 751; gray-purple, 49; black-red, 45. (a) What is the recombination frequency between these genes for body color and eye color? (b) Following up on this problem and problem 4, what fruit flies (genotypes and phenotypes) would you mate to determine the sequence of the body color, wing shape, and eye color genes on the chromosomes?

Solution

6. A space probe discovers a planet inhabited by creatures who reproduce with the same hereditary patterns as those in humans. Three phenotypic characters are height (T = tall, t = dwarf), hearing appendages (A = antennae, a = no antennae), and nose morphology (S = upturned snout, s = downturned snout). Since the creatures were not “intelligent” Earth scientists were able to do some controlled breeding experiments, using various heterozygotes in testcrosses. For a tall heterozygote with antennae, the offspring were tall-antennae, 46; dwarf-antennae 7; dwarf-no antennae 42; tall-no antennae 5. For a heterozygote with antennae and an upturned snout, the offspring were antennae-upturned snout 47; antennae-downturned snout, 2; no antennae-downturned snout, 48: no antennae-upturned snout 3. Calculate the recombination frequencies for both experiments.

Solution

7. Using the information from problem 6, a further testcross was done using a heterozygote for height and nose morphology. The offspring were tall-upturned nose, 40; dwarf-upturned nose, 9; dwarf-downturned nose, 42; tall-downturned nose, 9. Calculate the recombination frequency from these data; then use your answer from problem 6 to determine the correct sequence of the three linked genes.

Solution

8. Imagine that a geneticist has identified two disorders that appear to be caused by the same chromosomal defect and are affected by genomic imprinting: blindness and numbness of the limbs. A blind woman (whose mother suffered from numbness) has four children, two of whom, a son and daughter, have inherited the chromosomal defect. If this defect works like Prader-Willi and Angelman syndromes, what disorders do this son and daughter display? What disorders would be seen in their sons and daughters?

Solution

9. What pattern of inheritance would lead a geneticist to suspect that an inherited disorder of cell metabolism is due to a defective mitochondrial gene?

Solution

10. An aneuploid person is obviously female, but her cells have two Barr bodies. what is the probable complement of sex chromosomes in this individual?

Solution

11. Determine the sequence of genes along a chromosome based on the following recombination frequencies: A-B, 8 map units; A-C, 28 map units; A-D, 25 map units; B-C , 20 map units; B-D, 33 map units.

Solution

12. About 5% of individuals with Downs syndrome are the result of chromosomal translocation. In most of these cases, one copy of chromosome 21 becomes attached to chromosome 14. How does this translocation lead to children with Down syndrome?

Solution

13. Assume genes A and B are linked and are 50 map units apart. An individual heterozygous at both loci is crossed with an individual who is homozygous recessive at both loci. (a) What percentage of the offspring will show phenotypes resulting from crossovers? (b) If you did not know genes A and B were linked, how would you interpret the results of this cross?

Solution

14. In Drosophila, the gene for white eyes and the gene that produces “hairy” wings have both been mapped to the same chromosome and have a crossover frequency of 1.5%. A geneticist doing some crosses involving these two mutant characteristics noticed that in a particular stock of flies, these two genes assorted independently; that is they behaved as though they were on different chromosomes. What explanation can you offer for this observation?

Solution

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Campbell Chapter 14 Gen Prob 1

Molecular Genetics: Problem 1A man with hemophilia (a recessive , sex-linked condition has a daughter of normal phenotype. She marries a man who is normal for the trait. What is the probability that a daughter of this mating will be a hemophiliac? A son? If the couple has four sons, what is the probability that all four will be born with hemophilia?

Genotypes:

A man with hemophilia is XhY where h = hemophilia gene and H = the normal gene.
Any daughter with normal phenotype whose father has hemophilia will be a carrier.

Her genotype must be:

XhXH and NOT XHXH We can use a Punnett square to show the probability of a daughter or son having hemophilia.

daughter x normal manXhXH x XHY

A. If the daughter marries a normal male the probability of a daughter having hemophilia is zero.

B. About 50% of male children would have hemophilia (Boxes 2 and 4 above)

C. The probability that all 4 sons have inherited hemophilia would be: 1/2 x 1/2 x 1/2 x 1/2 or 1/16.

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