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Griffiths AJF, Miller JH, Suzuki DT, et al. An Introduction to Genetic Analysis. 7th edition. New York: W. H. Freeman; 2000.

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An Introduction to Genetic Analysis. 7th edition.

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Functional genomics

Large-scale sequence data are the beginning of functional genomics. The following sections show some of the analyses that can be performed to investigate function.

Characterize the proteome by ORF analysis

The genomic DNA sequence is analyzed by computer gene-prediction software that, among other things, examines each of the six reading frames of all sequences and searches for segments beginning with the translational start codon AUG and ending with a stop codon. Any open reading frames of at least 100 codons are candidates for genes. Most ORFs are completely novel, not corresponding to any familiar gene with alleles producing identifiable phenotypes. The ORFs can be analyzed for function initially by using the computer to search data bases to look for full or partial homology to known genes characterized in other organisms. The location, orientation, and clustering of ORFs also are important genomic information. Examples from Haemophilus and Saccharomyces are shown in Figures 14-21 and 14-22. A provisional proteome gene distribution can be deduced from such analysis, as shown in Figure 14-23. In higher eukaryotes, in which introns are common features of transcripts, predicting from ORF genomic DNA is more difficult.

Figure 14-21. The architecture of the genome of the bacterium Haemophilus influenzae, based on the complete genomic sequence reported in 1995.

Figure 14-21

The architecture of the genome of the bacterium Haemophilus influenzae, based on the complete genomic sequence reported in 1995. The circles have the following meanings, starting with the outer circle:⧫ Restriction sites and genomic positions, (more...)

Figure 14-22. The genetic landscape of chromosome 3 in yeast, determined by sequencing the entire chromosome.

Figure 14-22

The genetic landscape of chromosome 3 in yeast, determined by sequencing the entire chromosome. Genes previously detected from mutant phenotypes are shown in green. Open reading frames, which likely are protein-encoding genes, were detected by sequence analysis (more...)

Figure 14-23. Distributions of various categories of protein-encoding genes, estimated from currently known genes in the plant Arabidopsis thaliana.

Figure 14-23

Distributions of various categories of protein-encoding genes, estimated from currently known genes in the plant Arabidopsis thaliana.

Gene disruption knockouts

ORF function can be investigated by systematically knocking out the gene by in vitro mutagenesis and then looking for any possible mutant phenotype that might provide clues about function. This process is underway in the fully sequenced genomes. Interestingly, many knocked-out ORFs show no phenotypic effects. More than half of the predicted ORFs may fall into this category.

The study of gene interactions by the yeast two-hybrid system

This method investigates interaction with the use of a two-plasmid system in yeast. The basis for the test is the yeast GAL4 transcriptional activator. This protein has two domains, a DNA-binding domain and an activation domain, both of which must be in close juxtaposition in order for the protein to initiate transcription. A gene for one protein under investigation is spliced next to the GAL4 DNA-binding domain on one plasmid and acts as “bait.” On another plasmid a gene for another protein being tested is spliced to the activation domain; this protein is said to be the “target” (Figure 14-24). The two plasmids are then introduced into the same cell. One way of doing so is to mate haploid cells containing bait and target. The only way in which the GAL4 binding and activation domains can come together is if the bait and target proteins bind to each other, demonstrating a physical interaction. The two-hybrid system can be automated to facilitate large-scale hunting for protein interactions throughout the proteome.

Figure 14-24. The yeast two-hybrid system for detecting gene interaction.

Figure 14-24

The yeast two-hybrid system for detecting gene interaction. The system uses the binding of two proteins under test to restore the function of the GAL4 protein, which activates a reporter gene.

The study of developmental regulation by using DNA chips

DNA chips are about to revolutionize genetics in the same way that silicone chips revolutionized the computer industry. DNA chips are samples of DNA laid out in regimented arrays bound to a glass “chip” the size of a microscope cover slip.

One protocol is as follows. Robotic machines with multiple printing tips resembling miniature fountain pen nibs deliver microscopic droplets of DNA solution to specific positions (addresses) on the chip. The DNA is dried and treated so that it will bind to the glass. Thousands of samples can be applied to one chip. Commonly, the array of DNAs are known cDNAs from different genes. In principle, all the cDNAs of the entire genome could be arrayed on chips. The chips are exposed to a heterogeneous labeled cDNA sample made from total mRNA isolated at some specific stage of development. Fluorescent label is used, and the binding of the probe molecules to the glass chip is monitored automatically by laser beams. A typical result is shown in Figure 14-25a. In this way, the genes that are active at any stage of development or under any environmental condition can be assayed. Once again the idea is to identify protein networks that are active in the cell at any particular stage of interest. Figure 14-26 shows an example of a developmental expression sequence.

Figure 14-25. Fluorescence detection of binding to DNA microarrays: (a) Array of 1046 cDNAs probed with bone-marrow-based cDNA probe.

Figure 14-25

Fluorescence detection of binding to DNA microarrays: (a) Array of 1046 cDNAs probed with bone-marrow-based cDNA probe. Intensity of signal follows the colors of the spectrum with red highest and blue lowest. (b) Affymetrix GeneChip, a 65,000-oligonucleotide (more...)

Figure 14-26. Display of gene-expression patterns detected by DNA microarrays.

Figure 14-26

Display of gene-expression patterns detected by DNA microarrays. Each row is a different gene and each column a different time point. Red means active; green inactive. The four columns labeled +cyc are from cells grown on cycloheximide. (Mike Eisen and (more...)

Another protocol loads the chip with an array of oligonucleotides synthesized nucleotide by nucleotide on the chip itself (Figure 14-27). The glass is first covered with protecting groups that prevent DNA deposition. A mask is placed on the glass with holes corresponding to the sites of deposition. Then laser beams are shone onto the holes where synthesis is to begin. The light knocks off the protecting groups. Then the glass is bathed in the first nucleotide to be deposited. Each nucleotide carries its own protection group, which can be knocked off for the second round of deposition. Hence, by the sequential application of the appropriate masks and bathing sequences, arrays of different nucleotides can be built up. For studying genomic function, these oligonucleotides could be identifying sequences of genes, such as ESTs. As before, the completed array is bathed in fluorescent probe. Binding to an oligonucleotide array is shown in Figure 14-23b.

Figure 14-27. One method for the synthesis of a large array of oligonucleotides on a glass chip in situ.

Figure 14-27

One method for the synthesis of a large array of oligonucleotides on a glass chip in situ. Nucleotides are deposited one at a time at addresses activated by light shining through a pattern of holes in a mask. Each nucleotide carries a blocking group that (more...)

Note that these DNA array methods basically take an approach to genetic dissection that is an alternative to mutational analysis. Under either method, the goal is to define the set of genes or proteins that are important to any specific process under study. Traditional mutational analysis does this by amassing mutations that affect a specific process under study; chip technology does it by detecting the specific mRNAs that are transcribed during that process.

DNA chips can also be used to detect mutations. Oligonucleotides can be prepared that are complementary to all possible simple mutational changes in a genetic region under analysis. Alternatively, oligonucleotides complementary to all the known mutations in a human gene (such as a breast cancer gene) can be arrayed on the chip.

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2000, W. H. Freeman and Company.
Bookshelf ID: NBK22052

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