CHEM 527   Introductory Biochemistry
Problem Set 8a

Mutational Analysis

1. The a -chain of human hemoglobin is normally 141 amino acid residues long. Three unusual variants have been discovered in which the a -chain is longer than normal. Hb-Constant Spring and Hb-Icaria both have a -chains 172 residues long and differ from each other only by one amino acid at position 142. The first 141 residues are identical with the entire normal a -chain. The a -chain of Hb-Wayne is 146 residues long. The last 8 residues differ from both normal and Hb-Constant Spring a -chains. The amino acid sequences from residue 138 to 148 are presented below. Each of these inherited defects can be explained by single base mutational events.

Residue No.           138 139 140 141 142 143 144 145 146 147 148...

Normal                Ser-Lys-Tyr-Arg

Constant Spring       Ser-Lys-Tyr-Arg-Gln-Ala-Gly-Ala-Ser-Val-Ala-

Icaria                Ser-Lys-Tyr-Arg-Lys-Ala-Gly-Ala-Ser-Val-Ala-

Wayne                 Ser-Asn-Thr-Val-Lys-Leu-Glu-Pro-Arg

a. What is the termination codon for normal a -chain? Be specific as possible and explain your deduction.

b. From the above sequences it is possible to unambiguously deduce a 26-base sequence of normal a-chain mRNA. Write out 15 bases of this sequence extending 6 bases on either side of the normal termination codon.

c. Write out the base sequence of the sense strand of DNA coding for the mRNA in Part b.

d. Specifically what are the base changes that can explain each of the three hemoglobin variants? Can this be ascertained unambiguously?
 

2a. Baker's yeast, Saccharomyces cerevisiae, is a facultative anaerobe. What might be the phenotypic characteristics of a yeast defective in the gene for cytochrome c? (i.e. consider examples of carbon sources it could or could not grow on, the effect if any of O2, and end products of metabolism likely to be found in the medium). Give examples of other mutants would have similar phenotypes.

a. The N-terminal amino acid sequence of cytochrome c from yeast is:
    Thr-G1u-Phe-Lys-Ala-Gly-

    The absence of what feature suggests that the protein is modified subsequent to its synthesis on the ribosome?

    b. A series of mutants lacking cytochrome c activity were isolated. Most produced defective proteins which would crossreact with antibodies to cytochrome c. Of the following classes of mutants, which would produce crossreacting material (CRM), which would not, and which may or may not. (Assume a mutant protein greater than 60% like the wild type protein will crossreact).

i. deletion of cytochrome c gene

ii. point mutation resulting in a single amino acid substitution

iii. mutation in the termination codon

iv. mutation in the initiation codon

v. amber mutation

vi. frameshift mutation

 d. Mutants lacking CRM were screened for revertants having a functional cytochrome c. Most of the revertants had the N-terminal sequence of the wild type cytochrome c. There were, however, several revertants with altered N-terminal sequences.
 

Revertant         Frequency                              N-terminal Sequence
Wild type     21                 Thr-Glu-Phe-Lys-Ala-Gly-Ser-Ala-Lys..

    A          4                                 Ala-Gly-Ser-Ala-Lys..

    B         13         Met-Ile-Thr-Glu-Phe-Lys-Ala-Gly-Ser-Ala-Lys..

    C          4         Met-Leu-Thr-Glu-Phe-Lys-Ala-Gly-Ser-Ala-Lys..

    D          2         Met-Arg-Thr-Glu-Phe-Lys-Ala-Gly-Ser-Ala-Lys..

    E          1    (10%)Met-Val-Thr-Glu-Phe-Lys-Ala-Gly-Ser-Ala-Lys..
                    (90%)    Val-Thr-Glu-Phe-Lys-Ala-Gly-Ser-Ala-Lys..

What is the nature of the original mutant lacking CRM and revertant mutation in each case? (Refer to the genetic code)

e. With reference to Question 2d, what can be deduced about the specificity of the enzyme which modifies wild type cytochrome c?


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Last updated: 22 September 1999 by Hal White
Copyright 1999, Department of Chemistry and Biochemistry, University of Delaware, Newark, DE  19716