Ann Richmond's laboratory gains new insights into tumor growth and wound healing through studies of the "SOS gene"

Allison Byrum/Intern
Jan. 28, 2001

DNA

DNA (deoxyribonucleic acid) is the master molecule of heredity. It is made up of two strands linked together by four bases in the form of a double helix. Each strand of DNA is made up of a backbone and bases that are strung in a precise order. The two strands of DNA come together like a zipper. The teeth, or bases, on one side of the zipper match with the corresponding bases on the other side. The bases are the nucleic acids adenine (A), guanine (G), cytosine (C), and thiamine (T). Adenine and thiamine always pair together, as do guanine and cytosine. The four bases are the letters in the alphabet of life.

Transcription

In the process called transcription, the two strands of DNA unzip so the bases are exposed. By assembling bases in the same sequence as that of one of the DNA strands, the protein machinery within the cell makes RNA, or ribonucleic acid. RNA is made up of the same four bases as DNA (except thiamine, which is replaced by uracil (U), but it is single stranded. So RNA is a copy of half of the original DNA. Once the RNA is made, special proteins pare it down to the information-carrying sections, which are called exons. The sections that are cut out are called introns, or intervening sections. It is a process roughly analogous to editing video. Say a vacationer with a video camera shot a lot of video footage and wants to edit it into a short program to show friends and relatives. To do so, he must go through the footage and copy only the best scenes onto a new tape. Similarly, the cell’s machinery extracts only the sequences it needs to construct a given protein from the DNA and combines them in a temporary RNA copy, which is called messenger RNA, or mRNA.

Translation

Once messenger RNA is produced, translation begins. Translation is the process that produces a specific protein based on the blueprint contained in the mRNA. Proteins consist of chains of amino acids. Each amino acid is coded for by a sequence of three bases called a codon. For example, the codon with the sequence guanine (G), cytosine (C), adenine (A) codes for the amino acid alanine. Complicating the picture is the fact that several codons specify the same amino acid. Other codons mark the beginning and end of proteins: The codon AUG, for example, is called the start codon because it signals the beginning of a protein sequence. When an mRNA containing these instructions is released from the cell, it attaches to an organelle, called a ribosome, that acts as a microscopic protein factory. The ribosome reads each codon and attaches the correct amino acid to the growing protein molecule until it is completed.

NUCLEIC ACID BASES: adenine (A), guanine (G), cytosine (C), and thiamine (T)
(In messenger RNA thiamine is replaced by a fifth base, uracil (U).)

Protein characterization

To identify the protein MGSA, Richmond had to reverse the normal translation and transcription process. She began with the complicated process of purifying the protein from tumor cells. She then used several different procedures that separated proteins by their charge and size to isolate MGSA. Once she had enough purified MGSA, Richmond turned to a process called microsequencing to determine the protein’s primary structure . Proteins are made of strings of amino acids. To determine the order in which the amino acids are assembled, amino acids must be knocked free from the end of the protein one by one and the identity of each amino acid determined. When only a small amount of the protein is present, the amino acid sequence is determined on a micro-scale, hence the term: microsequencing. This provides the order of the amino acids. Since several codons code for each amino acid, however, Richmond was forced to turn to yet another technology to determine the order of the bases in the messenger RNA: a technology called molecular cloning from a cDNA library.

cDNA Libraries

In order to determine the structure of proteins, researchers are interested in the messenger RNA sequences that code for each amino acid. However, mRNA is unstable. It easily degrades under normal temperatures and conditions, making it difficult to work with directly. So researchers imitate nature by converting all the mRNA from a given source into DNA, which is extremely stable. Because these are copies of DNA, researchers call them cDNA libraries. The scientists use these libraries when they know portions of the amino acid sequence of a protein. From this information, they make a few matching codon sequences and then use this as a “hook” to catch the strands of DNA that contain matching sequences. Once they have retrieved the correct DNA sequence from the thousands contained in a cDNA library, they can obtain the sequence from the appropriate segment of the DNA and so nail down the gene’s exact sequence.

DNA extraction, digestion and PCR

Richmond’s team currently spends a portion of its time investigating the roles of different chemokines in processes such as wound healing and tumor growth. To do so, the researchers must know when the mice that they use as an animal model encode variant genes for specific chemokines. They do this by extracting some DNA from the animal and testing it to see if it has a given chemokine gene. First, a researcher removes a tiny bit of skin. Special chemicals are used to digest everything but the DNA. The DNA is then run through a process called a polymerase chain reaction. In the PCR process, the double-stranded DNA is heated just enough so that it opens up and can be copied. Researchers can regulate which sections of the DNA will be copied. By doing this repeatedly, a PCR machine can produce thousands of copies of a specific section of DNA. The multiplied DNA is then mixed with a loading dye and put onto one end of a gel, a material with the consistency of Jell-O that is spread about one-fourth-inch thick onto a sheet of hard plastic. Electricity is run through the gel in order to exert pressure on the DNA molecules. Smaller pieces of DNA move faster and so travel farther than the heavier ones. Once the gel has run for several minutes a fluorescent chemical called ethidium bromide, or EtBr, is added. EtBr mixes with the DNA and shows the DNA’s position when viewed under ultraviolet light. The test allows researchers to identify specific fragments of DNA.