H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003), страница 10

Once a smallamount of purified protein is obtained, antibodies to it canbe produced by methods discussed in Chapter 6. For a biochemist, antibodies are near-perfect tools for isolating largeramounts of a protein of interest for further analysis. In effect,antibodies can “pluck out” the protein they specifically recognize and bind from a semipure sample containing numerous different proteins. An increasingly common alternative isto engineer a gene that encodes a protein of interest with asmall attached protein “tag,” which can be used to pull outthe protein from whole cell extracts.Purification of a protein is a necessary prelude to studieson how it catalyzes a chemical reaction or carries out otherfunctions and how its activity is regulated.

Some enzymes aremade of multiple protein chains (subunits) with one chaincatalyzing a chemical reaction and other chains regulatingwhen and where that reaction occurs. The molecular machines that perform many critical cell processes constituteeven larger assemblies of proteins. By separating the individual proteins composing such assemblies, their individual catalytic or other activities can be assessed.

For example,purification and study of the activity of the individual proteins composing the DNA replication machine providedclues about how they work together to replicate DNA duringcell division (Chapter 4).The folded, three-dimensional structure, or conformation, of a protein is vital to its function. To understand the relation between the function of a protein and its form, weneed to know both what it does and its detailed structure.The most widely used method for determining the complexstructures of proteins, DNA, and RNA is x-ray crystallography.

Computer-assisted analysis of the data often permits thelocation of every atom in a large, complex molecule to be determined. The double-helix structure of DNA, which is keyto its role in heredity, was first proposed based on x-ray crystallographic studies. Throughout this book you will encounter numerous examples of protein structures as we zeroin on how proteins work.HomogenatBiochemistry Reveals the Molecular Structureand Chemistry of Purified Cell Constituentse1.4 • Investigating Cells and Their Parts345▲ FIGURE 1-22 Biochemical purification of a protein from acell extract often requires several separation techniques. Thepurification can be followed by gel electrophoresis of the startingprotein mixture and the fractions obtained from each purificationstep.

In this procedure, a sample is applied to wells in the top ofa gelatin-like slab and an electric field is applied. In the presenceof appropriate salt and detergent concentrations, the proteinsmove through the fibers of the gel toward the anode, with largerproteins moving more slowly through the gel than smaller ones(see Figure 3-32). When the gel is stained, separated proteins arevisible as distinct bands whose intensities are roughlyproportional to the protein concentration. Shown here areschematic depictions of gels for the starting mixture of proteins(lane 1) and samples taken after each of several purificationsteps. In the first step, salt fractionation, proteins thatprecipitated with a certain amount of salt were re-dissolved;electrophoresis of this sample (lane 2) shows that it containsfewer proteins than the original mixture. The sample then wassubjected in succession to three types of columnchromatography that separate proteins by electrical charge, size,or binding affinity for a particular small molecule (see Figure3-34).

The final preparation is quite pure, as can be seen from theappearance of just one protein band in lane 5. [After J. Berg et al.,2002, Biochemistry, W. H. Freeman and Company, p. 87.]Genetics Reveals the Consequencesof Damaged GenesBiochemical and crystallographic studies can tell us muchabout an individual protein, but they cannot prove that it isrequired for cell division or any other cell process. The importance of a protein is demonstrated most firmly if a mu-22CHAPTER 1 • Life Begins with Cellstation that prevents its synthesis or makes it nonfunctionaladversely affects the process under study.We define the genotype of an organism as its compositionof genes; the term also is commonly used in reference to different versions of a single gene or a small number of genesof interest in an individual organism.

A diploid organismgenerally carries two versions (alleles) of each gene, one derived from each parent. There are important exceptions, suchas the genes on the X and Y chromosomes in males of somespecies including our own. The phenotype is the visible outcome of a gene’s action, like blue eyes versus brown eyes orthe shapes of peas. In the early days of genetics, the locationand chemical identity of genes were unknown; all that couldbe followed were the observable characteristics, the phenotypes. The concept that genes are like “beads” on a long“string,” the chromosome, was proposed early in the 1900sbased on genetic work with the fruit fly Drosophila.In the classical genetics approach, mutants are isolatedthat lack the ability to do something a normal organism cando.

Often large genetic “screens” are done, looking for manydifferent mutant individuals (e.g., fruit flies, yeast cells) thatare unable to complete a certain process, such as cell divisionor muscle formation. In experimental organisms or culturedcells, mutations usually are produced by treatment with amutagen, a chemical or physical agent that promotes mutations in a largely random fashion. But how can we isolateand maintain mutant organisms or cells that are defective insome process, such as cell division, that is necessary for survival? One way is to look for temperature-sensitive mutants.These mutants are able to grow at one temperature, the permissive temperature, but not at another, usually higher temperature, the nonpermissive temperature.

Normal cells cangrow at either temperature. In most cases, a temperaturesensitive mutant produces an altered protein that works atthe permissive temperature but unfolds and is nonfunctionalat the nonpermissive temperature. Temperature-sensitivescreens are readily done with viruses, bacteria, yeast, roundworms, and fruit flies.By analyzing the effects of numerous different temperaturesensitive mutations that altered cell division, geneticists discovered all the genes necessary for cell division without knowinganything, initially, about which proteins they encode or howthese proteins participate in the process. The great power of genetics is to reveal the existence and relevance of proteins without prior knowledge of their biochemical identity or molecularfunction.

Eventually these “mutation-defined” genes were isolated and replicated (cloned) with recombinant DNA techniques discussed in Chapter 9. With the isolated genes in hand,the encoded proteins could be produced in the test tube or inengineered bacteria or cultured cells. Then the biochemistscould investigate whether the proteins associate with other proteins or DNA or catalyze particular chemical reactions duringcell division (Chapter 21).The analysis of genome sequences from various organisms during the past decade has identified many previouslyunknown DNA regions that are likely to encode proteins(i.e., protein-coding genes).

The general function of the protein encoded by a sequence-identified gene may be deducedby analogy with known proteins of similar sequence. Ratherthan randomly isolating mutations in novel genes, severaltechniques are now available for inactivating specific genesby engineering mutations into them (Chapter 9). The effectsof such deliberate gene-specific mutations provide information about the role of the encoded proteins in living organisms.

This application of genetic techniques starts with agene/protein sequence and ends up with a mutant phenotype;traditional genetics starts with a mutant phenotype and endsup with a gene/protein sequence.Genomics Reveals Differences in the Structureand Expression of Entire GenomesBiochemistry and genetics generally focus on one gene and itsencoded protein at a time. While powerful, these traditionalapproaches do not give a comprehensive view of the structure and activity of an organism’s genome, its entire set ofgenes.

The field of genomics does just that, encompassing themolecular characterization of whole genomes and the determination of global patterns of gene expression. The recentcompletion of the genome sequences for more than 80species of bacteria and several eukaryotes now permits comparisons of entire genomes from different species. The resultsprovide overwhelming evidence of the molecular unity of lifeand the evolutionary processes that made us what we are (seeSection 1.5). Genomics-based methods for comparing thousands of pieces of DNA from different individuals all at thesame time are proving useful in tracing the history and migrations of plants and animals and in following the inheritance of diseases in human families.New methods using DNA microarrays can simultaneously detect all the mRNAs present in a cell, thereby indicating which genes are being transcribed.

Such globalpatterns of gene expression clearly show that liver cells transcribe a quite different set of genes than do white blood cellsor skin cells. Changes in gene expression also can be monitored during a disease process, in response to drugs or otherexternal signals, and during development. For instance, therecent identification of all the mRNAs present in cultured fibroblasts before, during, and after they divide has given usan overall view of transcriptional changes that occur duringcell division (Figure 1-23). Cancer diagnosis is being transformed because previously indistinguishable cancer cellshave distinct gene expression patterns and prognoses (Chapter 23).