Genomics and Proteomics

Genomics and Proteomics

With more and more full DNA sequences becoming available, it is tempting to compare those sequences to see whether patterns emerge from genes that encode proteins with similar functions. The amount of data makes use of a computer essential for the process. Databases on genome and protein sequences are so extensive as to require information technology at its best to solve problems. Knowing the full DNA sequence of the human genome, for example, allows us to address the causes of disease in a way that was not possible until now. That prospect was one of the main incentives for undertaking the Human Genome Project. The website of the National Human Genome Research Institute, which is a part of the National Institutes of Health (NIH) has useful information; the URL for this site is http :// www .genome.gov.

A number of genomes are available online, along with software for sequence comparisons. An example is the material available from the Sanger Institute (http :// www .sanger.ac.uk). In November 2003, the researchers at this institute announced that they had sequenced 2 billion bases from the DNA of several organisms (human, mouse, zebrafish, yeasts, and the roundworm Caenorhabditiselegans, among others). If this amount of DNA were the size of a spiral staircase,it would reach from the Earth to the Moon.

A question implicit in the determination of the genome of any organism is that of assignment of sequences to the chromosome in which they belong. This is a challenging task, and only suitable computer algorithms make it possible. Once this has been achieved, one can compare genomes to see what changes have occurred in the DNA of complex organisms compared with those of sim-pler ones.

Beyond this application, challenging though it may be, lies the application to medicine, which is leading to a number of surprises. Two closely related genes (BRCA1 and BRCA2) involved in the development of breast cancer inter-act with other genes and proteins, and this is a topic of feverish research. The connection between these genes and a number of seemingly unrelated cancers is only starting to be unraveled. Clearly, there is need to determine not only the genetic blueprint but the manner in which an organism puts it into action.

The proteome is the protein version of the genome. In all organisms for which sequence information is available, proteomics (the study of interac-tions among all the proteins in a cell) is assuming an important place in the life sciences. If the genome is the script, the proteome puts the play on stage. The genetically determined amino acid sequence of proteins determines their structure and how they interact with each other. Those interactions determine how they behave in a living organism. The potential medical applications of the human proteome are apparent, but these have not yet been realized. Proteomic information does exist, for eukaryotes such as yeast and the fruit fly Drosophila melanogaster, and the methods that have been developed for thoseexperiments will be useful in unraveling the human proteome.

The Power of Microarrays—Robotic Technology Meets Biochemistry

Thousands of genes and their products (i.e., RNA and proteins) in a given living organism function in a complicated and harmonious way. Unfortunately, traditional methods in molecular biology have always focused on analyzing one gene per experiment. In the past several years, a new technology, called DNAmicroarray (DNA chip or gene chip), has attracted tremendous interest amongmolecular biologists. Microarrays allow for the analysis of an entire genome in one experiment and are used to study gene expression, the transcription rates of the genome in vivo. The genes that are being transcribed at any particular time are known as the transcriptome. The principle behind the microarray is the placement of specific nucleotide sequences in an ordered array, which then base-pair with complementary sequences of DNA or RNA that have been labeled with fluorescent markers of different colors. The locations where binding occurred and the colors observed are then used to quantify the amount of DNA or RNA bound. Microarray chips are manufactured by high-speed robotics, which can put thousands of samples on a glass slide with an area of about 1 cm². The diameter of an individual sample might be 200 μm or less. Several different methods are used for implanting the DNA to be studied on the chip, and many companies make microarray chips.

How do microarrays work?

Figure 13.30 shows an example of how microarrays could be used to determine whether a potential new drug would be harmful to liver cells. In Step 1, a microarray is purchased or constructed that has single-stranded DNA representing thousands of different genes, each applied at a specific spot on the microarray chip. In Step 2, different populations of liver cells are collected, one treated with the potential drug and the other untreated. The mRNA being transcribed in these cells is then collected. In Step 3, the mRNA is converted to cDNA. Green fluorescent labels are added to the cDNA from the untreated cells, and red fluorescent labels are added to the cDNA from the treated cells. In Step 4, the labeled cDNAs are added to the chip. The cDNAs bind to the chip if they find their complementary sequences in the single-stranded DNAs loaded on the chip. The expanded portion of Step 4 shows what is happening at the molecular level. The black sequences represent the DNA bound to the chip. Green sequences represent the cDNA from the untreated cells that bind to their target sequences. The red sequences are cDNA from treated cells. Some of the DNA sequences on the chip bind nothing. Some bind only the red sequences while others bind only the green ones. Some sequences on the chip bind both.

In Step 5, the chip is scanned and a computer analyzes the fluorescence. The results appear as a series of colored dots. A red dot indicates a DNA sequence on the chip that bound to the cDNA from the treated cells. This indicates an mRNA that was being expressed in treated cells. A green dot indicates RNA produced in untreated but not treated cells. A yellow dot would indicate a mRNA that was produced equally well in treated or untreated cells. Black spaces indicate DNA sequences on the chip for which no mRNA was produced in either situation. To answer the question about whether the potential drug is toxic to liver cells, the results from the microarray would be compared to controls run with liver cells and drugs known to be toxic versus those known to be nontoxic, as shown in Step 6.

Figure 13.31 shows the results of a study designed to scan cells from cancer patients and correlate microarray patterns with prognoses. The four different patterns are compared to the percentage of patients who developed metastases. Information like this could be critical to treatment of cancer. Doctors often have to choose between different strategies. If they had access to data like this from their patients, they would be able to predict the likelihood of the patient’s developing more serious forms of cancer, and thereby able to choose a more appropriate treatment.

Protein Arrays

Another type of microchip uses bound proteins instead of DNA. These protein arrays are based on interactions between proteins and antibodies . For example, antibodies to known diseases can be bound to the microarray. A sample of a patient’s blood can then be put on the microarray. If the patient has a particular disease, proteins specific to that disease bind to the appropriate antibodies. Fluorescently labeled antibodies are then added and the microarray scanned. The results look similar to the DNA microarrays discussed previously. Figure 13.32 shows how this would work to identify that a patient had anthrax. This technique is growing in popularity and power, but is limited by whether purified antibodies have been created for a particular disease.

Summary

As more DNA sequences become available, it becomes possible to com-pare those sequences. Of particular interest is any pattern that may emerge from genes that encode proteins with similar functions.

Important medical applications are emerging, and new methods are mak- ing it possible to analyze large quantities of data. Complete protein–protein interaction maps are now available for eukaryotes.

The proteome is the protein version of the genome. It refers to all the proteins being expressed in a cell. The study of proteomics is becoming very important in the life sciences.

A very powerful technique in vogue these days is the use of DNA or protein microchips. With these chips, thousands of samples of DNA or proteins can be applied and then checked for binding of biological samples.

The binding is visualized by using fluorescently labeled molecules and scanning the chip with a computer. The pattern of fluorescent labels then indicates which mRNA or proteins are being expressed in the samples.