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PBL Assignment - A Virtual Study of Mendel's Plant Height Alleles

200 points total

Mendel's discovery of the principles of genetics is the most important unifying theory in biology. These principles describe the nature of genes and how they genetically interact to control gene expression. Since Mendel's time, a great amount of research has led to our understanding of the structure and expression of genes. Genes are made of DNA. The DNA is transcribed into RNA, which in turn is a substrate for the translation process that makes proteins. The proteins can act as enzymes in a biochemical pathway, agents that control gene expression or as structural components of the cell. These proteins are directly responsible for the phenotype of the organism. A change in the DNA sequence of the gene can directly affect the function of the protein. These changes may include a nucleotide substitution, or the addition or deletion of nucleotides from the sequence. All can have direct effects on the expression of the protein. An altered protein structure can lead to a new phenotype. In genetic terminology, an individual with a new phenotype is called a mutant.

Specifically, we will investigate the differences in gene structure of the pea gibberellin 3- hydroxylase gene. This gene is responsible for plant height in pea (and other species). A specific form of gibberellic acid (GA), a plant hormone, is necessary for stem elongation. If the correct form of the hormone, GA1, is not present, the stem doesn't elongate, and the plant is shorter in stature. A functional version of gibberellin 3- hydroxylase is necessary to convert GA20 to GA1. The mutated form of the protein cannot make the conversion. If you remember, plant height was one of the seven traits Mendel studied. The gene responsible for this trait is called Le. You will use bioinformatic tools to learn the nature of the Le mutants.

Computer scientists have written programs that allow geneticists to study gene sequences. The most important programs align two sequences and permit a detailed study of their sequence differences. We will use a program, MultAlin, which is available over the Internet. To use the program, you simply paste the sequences you wish to compare into the "Sequence box", set a few parameters, and have the program align the sequences.

  • Open a separate WWW browser and go to the Multalin WWW site:

    Multalin WWW Site

  • Go to the linked file below, and copy the "Le" and "le-1" sequences, the first two sequences in the file. (Make sure you copy the first line of each sequence. This starts with the character ">". This FASTA header line tells the program that the sequence begins on the next line.) Then return to the browser containing the Multalin site and paste the sequences into the "Sequence box".

    Mendels Le Alleles

  • You now need to set specific program parameters. Under the "Optional Parameters" section, go to the "Symbol comparison Table" and select "DNA - 5 - 0". (Make sure you click on the WWW page before you go on or you will lose the selection you just made.) Now go further down the page to the "Presentation options" section and set the "Maximum line length" to "100". Now click on "Start MultAlin!" and study the output.

QUESTIONS:

1. Carefully go through the alignment and look for differences. Do you see any? What differences do you see?

2. The sequences you analyzed represent the mRNAs for each of the two alleles. (The information for the one intron is not included.) The sequences begin with the three base pair ATG start codon that encodes the amino acid methionine. Using this information, can you figure out the amino acid change that would result from this mutation? The Genetic Code is included below for reference.

Many mutants in nature are result of simple changes. But by no means is that the only type of change that could occur. Since Mendel's time other Le mutants have been discovered. In each case, the plant is shorter than the tall wild type plant. Some mutants (le-2) were actually derived from Mendel's original dwarf mutant plant. le-2 is even shorter than le-1. So the question is: What are the structural differences in the various mutants.

  • Return to the MultAlin WWW site. This time cut and past the following sequences (Le, le-1, le-2, le-3, le-3*, and led. (Don't add the last sequence, le-2*.) Set the program parameters as before, and run MultiAlin.

QUESTIONS

3. What differences do you see?

4. What specific amino acid changes do you predict for certain mutants?

5. Do you observe a new mutant changes? If so, describe those.

6. Can you determine which mutants are related?

7. What information supports your conclusion?

8. What differences do you see between le-1 and le-2? Remember these two mutants are related?

  • Using the Back button, return to the form page. Now add mutant le-2* to the bottom of the list of sequences in the sequence and again run the analysis.
QUESTIONS

9. What is different about this mutant compared to the others?

10. What other mutant appears to be closely related to le-2*?

The Genetic Code

  T C A G
T TTT Phe (F)
TTC "
TTA Leu (L)
TTG "		 TCT Ser (S)
TCC "
TCA "
TCG "		 TAT Tyr (Y)
TAC "
TAA Ter
TAG Ter		 TGT Cys (C)
TGC "
TGA Ter
TGG Trp (W)
C CTT Leu (L)
CTC "
CTA "
CTG "		 CCT Pro (P)
CCC "
CCA "
CCG "		 CAT His (H)
CAC "
CAA Gln (Q)
CAG "		 CGT Arg (R)
CGC "
CGA "
CGG "
A ATT Ile (I)
ATC "
ATA "
ATG Met (M)		 ACT Thr (T)
ACC "
ACA "
ACG "		 AAT Asn (N)
AAC "
AAA Lys (K)
AAG "		 AGT Ser (S)
AGC "
AGA Arg (R)
AGG "
G GTT Val (V)
GTC "
GTA "
GTG "		 GCT Ala (A)
GCC "
GCA "
GCG "		 GAT Asp (D)
GAC "
GAA Glu (E)
GAG "		 GGT Gly (G)
GGC "
GGA "
GGG "