DESIGNER GENES KEY

SSSS

By Andromeda215

This test will take 50 minutes in total and is conducted in stations. Pay attention to the top of the page, as it will tell you how many minutes are permitted for that specific station. Show all your work/ calculations. You are permitted Two non-graphing calculators and One 8.5” by 11” cheat sheet. Best of luck!

Team name: ______

Team number: C - ____

Participant names: ______

STATION 1: GENETIC CROSSES 5 MIN

You are a geneticist studying a eukaryotic, sexually reproducing, microbial species known colloquially as Goofy Goobers. We will be calling them Goobers for short. Goober colonies have been found to be either red, purple, blue, or white.

For 1-2, options are separated by slashes. Circle the most appropriate option.

1. Red is an example of a Character/Trait. 2. Colony color is an example of a Character/Trait. 3. Describe the molecular basis for dominance. In other words, what makes certain alleles appear to “cover up” the effects of another allele? Dominant alleles tend to code for proteins (Ex: presence of Rh protein, presence of more melanin in hair) Recessive tends to code for the absence of those proteins. (Ex: Blood type Rh- is just an absence of the Rh protein, alleles corresponding to light blonde hair codes for no/less melanin) Therefore, if a person is heterozygous for a gene, they will still produce the protein encoded by the dominant allele, so they will display the corresponding phenotype. 4. Define the term “True-Breeding”. Has been verified homozygous. 5. Define the term “Mendelian Trait”. Character that is controlled by 1 , and there are only 2 possible alleles (completely dominant & completely recessive) 6. State Mendel’s Law of Independent Assortment and explain why/how it is not always followed in biology. “The allele that a gamete receives for 1 gene does not influence alleles received for different genes”. Not always followed because of genetic linkage. It is statistically less likely for genes on the same chromosome to segregate than genes from different chromosomes. 7. Explain the difference between genotype and phenotype and state the relationship between these two terms. Genotype is a representation of alleles that an organism possesses (can be overall, or in reference to a single gene. Ex: Gg and GG are different genotypes, but they can produce the same phenotype. Phenotype describes how the alleles are expressed. An organism may express a dominant allele, but that doesn’t really tell you much about what that organism’s genotype may be (heterozygous or homozygous dominant) Genotype determines phenotype. 8. Explain the difference between cis and trans heterozygotes. Cis heterozygotes: AB/ab Trans heterozygotes: Ab/aB 9. Explain the difference between incomplete dominance and codominance on the protein level. Inc dominance: Because one recessive & one dominant allele is present, only some protein is made, which affects the phenotype. Phenotype will turn out to look like a mix between recessive and dominant. (if it were both dominant, more protein would be made, so wouldn’t get this phenomenon) Codominance: Two different dominant alleles are present. Each of those alleles creates a different protein, so both corresponding phenotypes are expressed. STATION 2: GENETIC DISEASE 5 MIN

Now you are a genetic counselor who moonlights as a lab technician.

10. Define an ideogram: A diagram of a , where chromosomes are grouped together based on position and ordered in decreasing size.

11. Describe the features that make a karyotype asymmetrical: Unusual centromere position, very large size difference between smallest and largest chromosomes

12. The human Y chromosome is closest in size to Chromosome #7. 13. Humans have 44 nuclear, autosomal chromosomes. 14. Chromosome 1 is: a. The physically largest chromosome. b. The chromosome with the largest region of protein-coding DNA. c. The physically smallest chromosome. d. The chromosome with the largest region of ncDNA. e. None of the above 15. Define “metacentric”: Centromere is approximately in the middle of the chromosome, making it look symmetrical. 16. Name the location where DNA is found in the mitochondria. matrix 17. Give an explanation as to why it is (generally) physically impossible for fathers to pass on mitochondrial DNA to their children. (Hint: in order to get full points, there is a specific gene that you must talk about) After sperm fertilizes egg, the mitochondria inside the sperm kill themselves, controlled by cps-6 gene.

18. Name the primary physiological process that human mtDNA makes possible. (Hint: specifically, mtDNA codes for 13 enzymatic subunits in order to carry out this process) Oxidative phosphorylation 19. In pedigrees, Roman numerals are used to denote generations. Arabic numerals are used to denote individuals in each generation. 20. Define “Euploidy”: Correct, complete set of chromosomes 21. Define “nondisjunction”. A mistake in chromosomal separation during cell division that results in daughter cells having an incorrect number of chromosomes. 22. If you track meiotic division from oogonium to the mature egg, there will be 3 polar body/bodies left at the end, which are a result of uneven cytoplasmic division. Two of the final four haploid cells have n + 1 chromosomes, and the other two have n – 1 chromosomes. State whether nondisjunction occurred. If it did, also state which state of meiosis it occurred in (be as specific as possible). Nondisjunction occurred during Anaphase I. STATION 3: GENETIC DISEASE (cont.) 15 MIN

Recap: Now you are a genetic counselor who moonlights as a lab technician.

You are consulting with a family who has been afflicted with Poppy syndrome.

23. There are two living grandparents, named Nadejda and Patrick. They are married to each other. Nadejda is affected, her husband is not. They have 3 children, and they were all affected. Their names are Rohan, Sandy, Fillmore. Rohan is the oldest, and Sandy is the middle child. Rohan has a wife who is not affected, and their three children were born in this order: daughter, son, daughter. None were affected. Sandy has an unaffected husband. Their three children were born in this order: daughter, son, son. All were affected. Fillmore has a wife who is not affected, and their three children were born in this order: son, daughter, son. None were affected. Draw the pedigree, and label generations/individuals properly.

24. Predict this disease’s mode of inheritance: mitochondrial 25. Not all affected display the same symptoms. For example, Sandy only needs to take a few medications daily to keep her symptoms under control, but her daughter is a permanent patient at the hospital. State the name of this phenomenon. Taking into account the mode of inheritance, explain why this happens: Variable expressivity. Just because a person is affected with the disease doesn’t mean that all/most mitochondria in their cells contain the mutation-causing allele. Those that are less affected probably have a lower percentage of faulty mitochondria in their cells, and those more affected have a higher percentage. Sandy’s daughter may have been more affected because during meiosis, the egg that she came from happened to get a relatively higher concentration of mitochondria as a result of random cytoplasmic splittage.

You have carried out some genetic tests for some newborns.

Baby #1:

Baby #2:

Baby #3:

Baby #4:

For 34-37, state the baby’s sex and the colloquial name of any identifiable disorders (if none found, write “Healthy”.) In either case, also state the cytogenetic notation.

26. Baby #1: Female, Cri-du-Chat syndrome. 46, XX, del(5p)

27. Baby #2: Male, healthy. 46, XY

28. Baby #3: Male, Klinefelter’s syndrome. 47, XYY

29. Baby #4: Female, Patau syndrome. 47, XX, +13

Now you are meeting with a boy and his family. The boy possesses severe intellectual disability and they want to figure out whether it is genetic. You conduct a basic karyotype, compile a pedigree, and test for trinucleotide repeats.

30. Describe the steps needed to conduct a standard karyotyping, starting with cell extraction and ending with visualization. 1. Blood draw/ bone marrow biopsy/ amniocentesis 2. Insert in phytohemagglutinin solution 3. Use colcemid 4. Insert in hypotonic KCl soln 5. Treat with alcohol & acetic acid 6. Put on glass slide 7. Stain with Giemsa dye

31. You interview the family. His mother is slightly affected, and his maternal grandfather is obviously affected. His father, maternal grandmother, and paternal grandparents are not affected. Construct the pedigree.

32. Using the pedigree and karyotype, predict whether this disorder is heritable. If so, state the mode of inheritance. Yes, X-linked dominant.

33. You test for trinucleotide repeats, and there are around 400 CGG repeats in the X chromosome. State whether this is significant. If so, state the disorder that you would diagnose him with. Significant. .

STATION 4: GENE STRUCTURE + GENE TRANSFER 10 MIN You are a geneticist who is trying to insert a bioluminescence operon into E. Coli. 34. Name an example of an operon that codes for bioluminescence: lux operon.

For 35-58, write down the definition of the word. 35. Cistron DNA/RNA section coding for a single polypeptide. 36. Open Reading Frame Protein-coding DNA section. 37. Constitutional Expression Gene is constantly expressed at some level in order for normal cell machinery to function.

38. Downstream In same direction RNA polymerase would go, transcribing from promoter to terminator sequence.

39. Operon Set of adjacent genes coding for proteins with a similar purpose, controlled by a single promoter & operator.

40. TATA box Eukaryotic TATA-binding protein bind site (binding induces formation of the PIC)

41. Pribnow box Prokaryotic. Initial site of double helix splittage after RNA polymerase binding.

42. -35 element Binds to RNA polymerase’s sigma subunit

43. Transcription Factor Sequence-specific DNA binding factor. Must contain a DNA Binding Domain. 44. Enhancer Promote transcription of distal genes through TF binding

45. Rho-dependent: System of transcription termination that involves Rho factor binding to newly transcribed mRNA.

46. Core promoter (Also state the function) Section of promoter between proximal & distal. Binds RNA polymerase

47. Proximal promoter (Also state the function) Part of promoter closest to ORF. General transcription factor bind site.

48. Distal promoter (Also state the function) Part of promoter closest to ORF. General transcription factor bind site. 49. Intragenic Region (also state domains of life where it is found) Noncoding sections of DNA found between exons Eukaryotes

50. Preinitiation complex (also state domains of life where it is found) 100 protein complex required for transcription Eukaryotes, Archaea

51. Sense strand Opposite of the template strand. Similar sequence to resulting mRNA.

52. Repressor Protein that can bind to the operator in order to prevent transcription

53. Repressible Molecule binds to activator/repressor in order to decrease transcription

54. Inducible Molecule binds to activator/repressor in order to increase transcription

55. Cis-regulatory elements Regulatory sequence on same chromosome as target gene

56. Trans-regulatory elements Regulatory protein transcribed from different chromosome from target gene

57. Selectable Marker Gene inserted into a plasmid that allows selection for bacteria that contain the plasmid. Ex: Antibiotic resistance gene.

58. Multiple Cloning Site Site on a plasmid with restriction sites for many different restriction enzymes STATION 5: GENE STRUCTURE (cont.) + GENE TRANSFER 10 MIN Recap: You are a geneticist who is trying to insert a bioluminescence operon into E. Coli. Let’s call this Operon A.

Let’s say you already have some plasmids that contain a gene coding for Ampicillin resistance, a Multiple Cloning Site, and Origin of Replication. 59. What is the function of a plasmid? Insertion of a foreign gene into a cell.

60. Why was it important to include an Ampicillin resistance gene in these plasmids? So that we can later select for bacteria that contain the plasmid. Not all bacteria will uptake the plasmid even after treatment, so want to make sure only the bacteria containing the plasmid survive (since they’re the only ones who might contain the bioluminescence genes).

61. Why is it important to include an Origin of Replication in these plasmids? In order to ensure plasmid survival once inserted.

The MCS contains restriction sites for restriction enzymes EcoR1, Hind III, Bam HI, and Sma I. 62. Why is it useful for the MCS to contain so many restriction sites? Offers many options.

63. What nuclease type do all these enzymes fall under? Endonuclease

64. Explain how restriction enzymes get their name. From the organism they were first found in.

65. EcoR1, Hind III, and Bam HI produce sticky ends. Explain what this means. Don’t produce blunt cuts → after cutting, produces 2 separate molecules, both with 5’ or 3’ overhangs.

66. When constructing a plasmid, why is it ideal to use restriction enzymes that produce sticky ends? Ensures that gene is inserted in the correct orientation.

67. You decide to use Bam HI to insert your plasmid. This is the restriction site:

Therefore, the promoter end of the inserted operon will start with the sequence 5’GATC3’. The 3’ UTR of the inserted operon will end with the sequence 3’CTAG5’. 68. Describe the 3 main steps you would use in order to insert the operon into the plasmid. 1. Insert Bam HI so that plasmid gets cut in the right spot 2. Introduce copies of operon into solution 3. Insert DNA Ligase

69. Next, we insert the plasmids into the E. Coli. This process is called bacterial transformation. 70. There are several methods of inserting the plasmids into the bacteria. For each of the methods below, explain how each of them work: a. Calcium chloride treatment: Promotes plasmid binding to cell membrane

b. Heat shock: Creates pores in bacterial cell membrane

c. Electroporation: Also creates pores in bacterial cell membrane.

Now you transfer the E. Coli into petri dishes. All of them contain nutrient broth. - 1 dish contains tetracycline - 1 dish contains ampicillin - 1 dish contains ampicillin + tetracycline - 1 dish contains no antibiotics. 71. State which dishes you expect the E. Coli will survive in. Dish with no antibiotics & dish that contains only ampicillin

You notice that even after selecting for E. Coli that contain the plasmid, only about 10% of the colonies are bioluminescent. 72. Give an explanation as to why this happened (hint: it was an experimental design flaw, not a freak accident) All the plasmids contain genes for ampicillin resistance but of those, not all of them actually uptook the bioluminescence operon. You did not select for only plasmids that contain the target operon.

73. You took the Operon A from another type of bacteria. You didn’t have to extensively process those genes in order for this experiment to succeed. Give at least 3 reasons why this would be impossible if the donor sequence was from a human. 1. Different promoters in eukaryotes vs prokaryotes 2. Prokaryotes don’t have machinery to handle introns 3. Lack of Shine-Dalgarno sequence

STATION 6: DNA REPLICATION 5 MIN 74. Explain the semiconservative, dispersive, and conservative models of DNA replication, and state which one is accurate. Semiconservative: Each strand of the parent molecule is a template for a new daughter strand. Conservative: One parent molecule begets 1 daughter molecule made completely from the parent molecule + another daughter molecule with completely “new” nucleotides. Dispersive: Daughter molecules contain random streaks of “new” and parent molecules.

75. The origin of replication tends to be rich in nucleotides containing which type of bases? Explain why. A & T. There are only 2 hydrogen bonds between these two when base-paired, so they’re easier to tear apart (in order to create a replication fork).

76. True or false (circle): There is only one origin of replication in a prokaryotic chromosome. 77. True or false (circle): There is only one origin of replication in a eukaryotic chromosome. 78. DNA polymerase adds nucleotides in the 5’ to 3’ direction. 79. DNA polymerase also requires a free OH group on the preceding 3’ nucleotide in order to extend the molecule. 80. The lagging strand is created in small 100-200bp sections, while the leading strand is created continuously. Explain why this happens. Because a DNA molecule is antiparallel (each strand runs in opposite directions), but DNA polymerase can only construct in 1 direction. Therefore, one strand (leading) can be constructed continuously in the direction of the replication fork, but because of how DNA polymerase works, the other strand (lagging) is created in the direction away from the replication fork, so it can only be created in smaller chunks.

81. Define “Hayflick limit”. Number of divisions a cell can go through before it becomes senescent.

82. DNA polymerases create phosphodiester bonds.

83. Endonucleases break phosphodiester bonds.

84. Helicases break hydrogen bonds.

85. Explain why DNA-binding proteins tend to bind on the major grooves. More space to bind.

86. Give 3 reasons why DNA is arranged in a double helix: 1. Nitrogen bases are hydrophobic, so want to limit as much contact with water (physiological environment) as possible. 2. Biologically, it’s really important for DNA to not be exposed to mutagens that could be present in solution. 3. Only certain bond angles are permitted between the sugar and phosphate.

87. Define “purine” Nitrogenous compound containing 2 rings (pyrimidine & imidazole)

88. Which property allows DNA molecules to move across the gel during gel electrophoresis? Negative charge.

89. Explain the roles of exonucleases in eukaryotic DNA replication. Shaves off incorrectly added nucleotides during proofreading. Shaves off the ends of chromosomes (because of the limitations caused by having a linear chromosome)

90. Explain the role of Topoisomerase. Relieves winding tension ahead of replication fork.

91. DNA Gyrase is a special type of Topoisomerase. Explain why this enzyme is so necessary for bacterial DNA replication. Only type of Topoisomerase that can relax positively supercoiled DNA.

92. In eukaryotes, nuclear DNA replication occurs during which stage of the cell cycle? S phase (Interphase)

93. In eukaryotes, organelle DNA replication occurs during which stage of the cell cycle? All stages

94. In prokaryotes, when does plasmid replication occur? At any time

95. In prokaryotes, when does chromosomal DNA replication occur? Beginning of binary fission.