基因工程與原理

1. 基因工程與原理 GENE CLONING & DNA ANALYSIS T. A. BROWN Chapter 10 Studying Genomes

2. 12 Studying Genomes 12.1 Genome annotation 12.1.1 Identifying the genes in a genome sequence Searching for open reading frames Simple ORF scans are less effective at locating genes in eukaryotic genomes Gene location is aided by homology searching Comparing the sequences of related genomes 12.1.2 Determining the function of an unknown gene Assigning gene function by experimental analysis requires a reverse approach to genetics Specific genes can be inactivated by homologous recombination 12.2 Studies of the transcriptome and proteome 12.2.1 Studying the transcriptome Studying a transcriptome by sequence analysis Studying transcriptomes by microarray or chip analysis 12.2.2 Studying the proteome Separating the proteins in a proteome Identifying the individual proteins after separation 12.2.3 Studying protein–protein interactions Phage display The yeast two hybrid system

3. Post-genomic or Functional genomics 12.1 Genome annotation Bioinformatics Yeast Saccharomyces cerevisiae/ 6000 genes/ 2400 genes whose functions were unknown (orphans) 12.1.1 Identifying the genes in a genome sequence Searching for open reading frames (ORFs) Initiation codon (usually but not always ATG) Termination codon (TAA, TAG or TGA in most genomes) Figure 12.1 A double-stranded DNA molecule has six reading frames.

4. ORF scanning • Each termination codon will appear once every 43 = 64 bp. • The ORFs should not be longer than 30-40 codons. The average lengths of genes are 317 codons for E. coli., 483 codons for S. cerevisiae, and 450 codons for humans. Figure 12.2 The typical result of a search for ORFs in a bacterial genome. The arrows indicate the directions in which the genes and spurious (假的) ORFs run.

5. Simple ORF scans are less effective at locating genes in eukaryotic genomes There is more intergenic DNA and introns in eukaryotic genomes. Figure 12.3 ORF scans are complicated by introns. The nucleotide sequence of a short gene containing a single intron is shown. The correct amino acid sequence of the protein translated from the gene is given immediately below the nucleotide sequence, using the single-letter amino acid abbreviations. In this sequence the intron has been left out, because it is removed from the transcript before the mRNA is translated into protein. In the lower line, the sequence has been translated without recognizing that an intron is present. As a result of this error, the amino acid sequence appears to terminate within the intron.

6. Codon bias: leucine is specified by six codons (TTA, TTG, CTT, CTC, CTA and CTG), but in human genes leucine is most frequently coded by CTG. Exon-intron boundaries Figure 12.4 The consensus sequences for the upstream and downstream exon–intron boundaries of vertebrate introns. Py = pyrimidine nucleotide (C or T), N = any nucleotide. The arrows indicate the boundary positions. Upstream regulatory sequences: recognition signals for the DNA binding proteins/ variable CpG islands: 1 kb upstream of many genes (40-50%)

7. Gene location is aided by homology searching Figure 12.5 Homology between two sequences that share a common ancestor. The two sequences have acquired mutations during their evolutionary histories but their sequence similarities indicate that they are homologs.

8. Nucleotide or amino acid sequence? Figure 12.6 Lack of homology between two sequences is often more apparent when comparisons are made at the amino acid level. Two nucleotide sequences are shown, with nucleotides that are identical in the two sequences given in red and nonidentities given in blue. The two nucleotide sequences are 76% identical, as indicated by the asterisks. This might be taken as evidence that the sequences are homologous. However, when the sequences are translated into amino acids, the identity decreases to 28%, suggesting that the genes are not homologous, and that the similarity at the nucleotide level was fortuitous. Identical amino acids are shown in brown, and non-identities in green. The amino acid sequences have been written using the one-letter abbreviations. Basic local alignment search tool (BLAST)

9. Comparing the sequences of related genomes Homology analysis/ comparative genomics Synteny: conservation of gene order Figure 12.7 Using comparisons between the genomes of related species to test the authenticity of a short ORF. In this example, the questionable ORF is not present in the related genome and so is probably not a real gene.

10. 12.1.2 Determining the function of an unknown gene Assigning gene function by experimental analysis requires a reverse approach to genetics Figure 12.8 Forward and reverse genetics.

11. Specific genes can be inactivated by homologous recombination Figure 12.9 Gene disruption by homologous recombination.

12. Figure 12.10 The use of a yeast deletion cassette. Knockout mouse: the vector carrying the deletion cassette is microinjected into an embryonic stem cell.

13. 12.2 Studies of the transcriptome and proteome 12.2.1 Studying the transcriptome Studying a transcriptome by sequence analysis AluI Bsm FI Figure 12.11 Serial analysis of gene expression (SAGE).

14. Studying transcriptomes by microarray or chip analysis Figure 12.12 Microarray analysis. The microarray shown here has been hybridized to two different cDNA preparations, each labelled with a fluorescent marker. The clones which hybridize with the cDNAs are identified by confocal microscopy.

15. Figure 12.13 A DNA chip. A real chip would carry many more oligonucleotides than those shown here, and each oligonucleotide would be 20–30 nucleotides in length.

16. 12.2.2 Studying the proteome (proteomic) Separating the proteins in a proteome Protein profiling: to identify the constituents of a proteome Two techniques: protein electrophoresis and mass spectrometry Isoelectric focusing(pH) and SDS electrophoresis(M.W. or size) Isoelectric point: the position in the gradient where its net charge is zero. Figure 12.14 Two-dimensional gel electrophoresis.

17. Identifying the individual proteins after separation Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) Trypsin: cleaves proteins immediately after Arg or Lys. Figure 12.15 The use of MALDI-TOF in mass spectrometry. Ionized peptides are injected into the mass spectrometer (a) and their mass-to-charge ratios measured and displayed as a spectrum (b).

18. Isotope coded affinity tag (ICAT) Deuterium, 2H or D Figure 12.16 Analyzing two proteomes by ICAT. In the MALDI-TOF spectrum, peaks resulting from peptides containing normal hydrogen atoms are shown in blue, and those from peptides containing deuterium are shown in red. The protein under study is approximately 1.5 times more abundant in the proteome that has been labeled with deuterium.

19. 12.2.3 Studying protein–protein interactions Phage display Figure 12.17 Phage display. (a) Display of proteins on the surface of a recombinant filamentous phage. (b) The gene fusion used to display a protein. (c) One way of detecting interactions between a test protein and a phage from within a display library.

20. The yeast two hybrid system Figure 12.18 The yeast two hybrid system. (a) A pair of transcription factors that must interact in order for a yeast gene to be expressed. (b) Replacement of transcription factor 1 with the hybrid protein 1* abolishes gene expression as 1* cannot interact with transcription factor 2. (c) Replacement of transcription factor 2 with the hybrid protein 2* restores gene expression if the hybrid parts of 1* and 2* are able to interact.