Comprehensive Molecular Biology (LC4)

Many changes in the structure of hemoglobin have arisen by mutations. About one person in 100 carries a mutant hemoglobin gene, and these individuals have abnormal hemoglobin molecules in their blood. One of the most common abnormal hemoglobins is hemoglobin S, which causes sickle cell anemia. When the gene for hemoglobin S is inherited from both parents, all of the hemoglobin in the circulation is hemoglobin S and the individual suffers from severe anemia.

A first step in characterizing a protein often involves determining its molecular weight. From this information, different proteins may be compared and the number of amino acid residues in a protein can be determined. Here, students determine the molecular weight of two unknown proteins by comparing their electrophoretic migration with the migration of standard proteins. The protein standards and unknowns have been pre-stained so that your students can follow their progress during the separation as shown.

The Western blotting procedure is rapidly replacing conventional methods for identifying and characterizing specific proteins in complex protein mixtures. This technique is used extensively for this purpose in the research laboratory and is increasingly being used in diagnostic medicine for detecting proteins of disease agents such as the structural core proteins of the AIDS virus. Here, students will perform this technique to examine the evolutionary distance between different mammals.

Electrophoresis in agarose gels is the most common method used for determining the size of DNA molecules. In this introductory exercise, students determine the length of an unknown DNA molecule by comparing its electrophoretic mobility with six DNA molecules of known size as shown.

Plasmids are small circular DNA molecules found in most bacteria. A plasmid can exist in different structural states and these states can be distinguished by their migration on agarose gels. In this experiment, students study these structures using enzymes and electrophoresis and show that the structures are interconvertible. This exercise provides an introduction to higher-order DNA structure which is thought to be important in the control of transcription and replication in bacteria and, perhaps, in higher organisms as well.

The history of evolution is recorded in the genomes of present-day organisms and enlightened guesses of evolutionary events can be made by comparing DNA sequences in different species. Evidence drawn from this approach has led to the construction of family trees of organisms that agree remarkably well with those obtained from more traditional procedures. In fact, on a number of occasions, comparative DNA sequence analysis has been used to clarify and expand on phylogenetic relationships that were derived from classical studies.

The concept that different enzymes are found in different tissues, cell types, and cell organelles is illustrated in this multipart exercise where students use contemporary techniques to localize specific enzymes in cells and tissues. Students are introduced to enzyme cytochemistry in an experiment on the germinating corn seed where they show that peroxidase is produced by the aleurone, a cell layer that surrounds the endosperm. They then characterize the subcellular distribution of peroxidase in giant onion epithelial cells and show that the enzyme resides in the cell wall.

The exercise is introduced with a discussion of observations that were made in the early nineteenth century. Namely, that biological stains often show specific affinity for particular subcellular components. With this information, students use differential staining procedures to identify nuclear and cytoplasmic components in cells from thymus and onion root tip. In the second part of the exercise, cell nuclei are isolated from thymus tissue and their purity assessed by staining techniques.