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Biological Background

Cells represent a fundamental level of organization in all eukaryotic organisms (i.e., plants and animals). Simply, a eukaryotic cell is a membrane-bounded compartment containing the molecules and sub-compartments necessary to carry out a particular function. Note the number of sub-compartments and the complexity of their organization even in the simplified representation of a eukaryotic cell in Figure 1.1. In spite of their diversity and complexity, however, all cells contain the same basic components: DNA, RNA, and protein.


  
Figure 1.1: A simplified cartoon depicting the basic components of a eukaryotic cell. Reprinted from MOLECULAR CELL BIOLOGY by Lodish et al. ©1986, 1990, 1996 by Scientific American Books, Inc. Used with permission by W.H. Freeman and Company.

All information necessary for the functions of a cell to be carried out is contained in its DNA. A molecule of DNA is simply a long double helix composed of four basic units called nucleotides. The human genome, for instance, is a DNA sequence which is some three billion nucleotides long. DNA does not carry out cellular processes on its own, but instead encodes proteins that do. Each protein required by the cell is encoded in a stretch of DNA called a gene. Like DNA, proteins are all made from sequences of basic building blocks. Instead of the four nucleotides of DNA, proteins are made of strings of twenty amino acids. Amino acids are each chemically and structurally unique so that when arranged in long chains and folded properly, they constitute the proteins that provide all of the functionality needed by the cell. Each of the cellular compartments in Figure 1.1 contains many proteins, some unique to that compartment, some not. The combination of proteins in a particular compartment account for the structure, function and localization of that compartment.

For a cell to produce a protein, the corresponding DNA sequence (gene) is first transcribed into a molecule of RNA. RNA, like DNA, consists of four nucleotides arranged in a sequence. Each successive triplet of nucleotides in the RNA sequence codes for a single amino acid in the protein. Through a process known as translation, the RNA sequence is converted into an amino acid sequence and therefore a protein. The protein-coding portion of a typical mammalian gene is 2000 to 3000 nucleotides long, corresponding to a protein length of 700-1000 amino acids.

This simple overview of protein production does not begin to address the complexity of cellular functions that are carried out by those proteins. Suffice it to say that the entire field of cell biology is devoted to elucidating these details! Two areas of investigation relevant to the work described below are genomics (the study of genomes) and proteomics (the study of proteomes).

Genomics was born out of the ability to sequence the molecules of DNA in cells. It represents a significant advance because it is one of the first areas of biology to which quantitative analysis has been applied. For some time now it has been possible to rigorously compare DNA sequences (e.g., genes) to one another with the intent of placing a number on the degree to which two sequences are similar. The motivation for this analysis is that genes with similar sequence tend to code for proteins with similar structure and function. From this it follows that one can make a prediction about the structure and function of a new protein based on the similarity of its gene sequence to the gene sequence of proteins that have already been characterized. An important milestone in biology and the current focus of much of the work in genomics is obtaining the complete DNA sequence of the human genome, i.e., the Human Genome Project (http://www.ornl.gov/hgmis/). A complete DNA sequence does not correspond to complete knowledge regarding the many proteins it encodes, however. The field of proteomics has evolved to provide this information.

Although a protein starts out as a sequence of amino acids, it is quickly folded into a complex three dimensional structure (see Figure 1.2). From this structure, a protein derives its unique properties. A protein's amino acid sequence can provide some information about its structure and function, but complex studies involving a variety of disciplines including crystallography, nuclear magnetic resonance (NMR) spectroscopy, and biochemistry are required to properly assign structure and function to a protein. These studies are not yet as `high-throughput' as DNA sequencing, but there is a growing realization that additional information about each protein will be necessary to complement the raw sequence that will be the product of the Human Genome Project.


    
Figure 1.2: Space-filling models of a single molecule of human hemoglobin (A), and a portion of a mouse antibody like the one used in the experiments described below (B), where each `ball' represents an atom. Note that the linear sequences of amino acids are folded into complex, compact structures. These images were generated by a combination of the POVChem and POVRay programs and Protein Data Bank (PDB) files obtained from the Research Collaboratory for Structural Bioinformatics (http://www.rcsb.org).
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Even after every gene is sequenced and the structure and function of every protein has been determined, the monumental task of understanding how it all works together inside a cell will still exist. A partial list of the cell functions into which each protein must be placed includes: 1) the general category of metabolism - how does a cell acquire and use energy? 2) locomotion - how do cells change shape and move from one place to another? 3) cell signalling - how does a cell receive input from its environment and how do cells communicate with one another? 4) intracellular transport - how do cells move material from one place to another internally? 5) reproduction - how do cells proliferate?

One aspect of proteins that is not currently being adequately addressed is their subcellular localization (i.e., where within a cell does each protein carry out its function?). To illustrate the concept of subcellular localization, Figure 1.3 includes two different images of the same cell. Part A is a transmitted-light image showing the full extent of the cell. Part B is a fluorescence image (see Section 1.6 for a discussion of fluorescence) that depicts only the localization of a particular protein, in this case one that is found in the mitochondria. Localization information is important because it provides a context for a protein's structural and functional information. For example, two proteins that possess similar structure and function may in fact be found in distinct compartments within the cell and therefore may be involved in unrelated cellular processes. The work described below is the first of which we are aware that addresses the subcellular localization of proteins in a quantitative manner.


  
Figure 1.3: Images of the same cell collected using a transmitted light mode (A - Differential Interference Contrast), and a fluorescence mode (B). The fluorescence in B shows the localization of a mitochondrial protein. Scale bar $= 10\mu$m
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Next: Goals Up: Introduction Previous: Introduction
Copyright ©1999 Michael V. Boland
1999-09-18