Biochemical Genetics Lab

Some 150 years ago, Mendel carried out a series of remarkable experiments in a small plot of a Czech Monastery (it’s really small--I have seen it). One of his experiments depended on the fact that the dry seeds of some pea varieties are always round and smooth, while the dry seeds of another variety are always wrinkled. What were the results of crossing these two varieties and then again crossing their offspring?

Parents: Round X Wrinkled (P generation)

“Children:” All Round X All Round (F₁—first filial—crossed to each other)

“Grandchildren:” 76 Round and 24 wrinkled (F₂

His interpretation of such results gave birth to the concept of the gene.

Mendel didn’t know how genes exerted their effects. Now we know that they often do so by coding for specific enzymes, which in turn catalyze chemical reactions. Round seeds are round because they have more amylopectin (a kind of starch) than wrinkled seeds have, and hence they do not collapse and wrinkle up when they lose water. Amylopectin can be produced from amylose. The amylose→amylopectin reaction in turn is catalyzed by an enzyme, Starch Branching Enzyme 1 (SEB1). SEB1 is more efficient in round seeds. SEB1 thus produces more amylopectin in round seeds, supporting their more rigid structure.

The key 2 reactions we’d rely on today are:

Glucose-1-Phostphate → amylose→ amylopectin

Unfortunately, the two starches are not readily distinguishable with the iodine test we’ll use, but we’ll do our best! Amylopectin gives a light, reddish purple color, and amylose yields a darker, almost black, blue color.

Round seeds have about 4 times as much amylopectin as wrinkled seeds, but only about ½ as much amylose. The enzyme that converts G-1-P to amylose is called starch synthetase (SS).

In today’s lab, we’ll isolate (they will be in the centrifuged supernatant) the 2 enzymes from powdered wrinkled seeds and from powdered round seeds, respectively, and add to each extract G-1-P. We’ll then let the starch-producing reaction proceed for fixed time intervals (0, 10, 20 . . . 60 min; see Table 1, p. 115). At each fixed interval, we’ll carry out the iodine test for polysaccharides. Our expectations:

1. It’s possible that wrinkled seeds contain more amylose because they have more of the enzyme (SS) that produces this starch. If so, the wrinkled-seed extract will contain more SS, yielding a darker color with iodine test in the early reaction time intervals (e.g., after 10 minutes).
2. The SEB1 enzyme, on the other hand, is more efficient in round seeds, and might convert the round-seed preparation more rapidly to amylopectin, thus gradually making, in the later time intervals, the wrinkled-seed extract darker in the iodine test than in the round-seed extract.