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This text discusses the transformation of bacteria through DNA and the groundbreaking Avery, MacLeod, and McCarty Experiment. It also explores gene knockouts and transgenics using ES cells and the Cre/lox system.
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DNA Mediates Transformation Convert IIR to IIIS By DNA?
Avery MacLeod and McCarty Experiment Circa 1943
DNAse activity + means that activity is present All RNA gets degraded during enzyme preparation
3.12 Gene Knockouts and Transgenics • transgenics – Organisms created by introducing DNA prepared in test tubes into the germline. • The DNA may be inserted into the genome or exist in an extrachromosomal structure. Figure 03.27: Transfection can introduce DNA directly into the germline of animals. Photo reproduced from P. Chambon, Sci. Am. 244 (1981): 60-71. Used with permission of Pierre Chambon, Institute of Genetics and Molecular and Cellular Biology, College of France.
3.12 Gene Knockouts and Transgenics • Embryonic stem (ES) cells that are injected into a mouse blastocyst generate descendant cells that become part of a chimeric adult mouse. • When the ES cells contribute to the germline, the next generation of mice may be derived from the ES cell. • Genes can be added to the mouse germline by transfecting them into ES cells before the cells are added to the blastocyst.
3.12 Gene Knockouts and Transgenics Figure 03.29: ES cells can be used to generate mouse chimeras.
3.12 Gene Knockouts and Transgenics • An endogenous gene can be replaced by a transfected gene using homologous recombination. • The occurrence of successful homologous recombination can be detected by using two selectable markers, one of which is incorporated with the integrated gene, the other of which is lost when recombination occurs.
3.12 Gene Knockouts and Transgenics • The Cre/lox system is widely used to make inducible knockouts and knock-ins. • knockout – A process in which a gene function is eliminated, usually by replacing most of the coding sequence with a selectable marker in vitro and transferring the altered gene to the genome by homologous recombination. • knock-in – A process similar to a knockout, but more subtle mutations are made.
3.12 Gene Knockouts and Transgenics Figure 03.31: The Cre recombinase catalyzes a site-specific recombination between two identical lox sites, releasing the DNA between them.
4.1 Introduction • interrupted gene – A gene in which the coding sequence is not continuous due to the presence of introns. • primary (RNA) transcript – The original unmodified RNA product corresponding to a transcription unit. • RNA splicing – The process of excising introns from RNA and connecting the exons into a continuous mRNA.
4.1 Introduction • intron – A segment of DNA that is transcribed, but later removed from within the transcript by splicing together the sequences (exons) on either side of it. • mature transcript – A modified RNA transcript. Modification may include the removal of intron sequences and alterations to the 5′ and 3′ ends.
Figure 04.01: Interrupted genes are expressed via a precursor RNA.
Logo for a) Splice acceptor b) Splice Donor c) Initiator Met
AG/GT CAG/NT exon 1 intron 1 exon 2 4 3 2 1 1 2 3 4 5 6 9 8 7 6 5 4 3 2 1 1 2
4.2 An Interrupted Gene Consists of Exons and Introns • Introns are removed by RNA splicing, which occurs in cis in individual RNA molecules. • Mutations in exons can affect polypeptide sequence; mutations in introns can affect RNA processing and hence may influence the sequence and/or production of a polypeptide. Figure 04.02: Exons remain in the same order in mRNA as in DNA, but distances along the gene do not correspond.
4.3 Exon and Intron Base Compositions Differ • The four “rules” for DNA base composition are the first and second parity rules, the cluster rule, and the GC rule. • Exons and introns can be distinguished on the basis of all rules except the first. • The second parity rule suggests an extrusion of structured stem-loop segments from duplex DNA, which would be greater in introns. • The rules relate to genomic characteristics, or “pressures,” that constitute the genome phenotype.
4.4 Organization of Interrupted Genes May Be Conserved • Introns can be detected when genes are compared with their RNA transcription products by either restriction mapping, electron microscopy, or sequencing. • cDNA – A single-stranded DNA complementary to an RNA, synthesized from it by reverse transcription in vitro. Figure 04.03: Comparison of the restriction maps of cDNA and genomic DNA for mouse β-globin.
4.4 Organization of Interrupted Genes May Be Conserved • The positions of introns are usually conserved when homologous genes are compared between different organisms. • The lengths of the corresponding introns may vary greatly. • Introns usually do not encode proteins.
Figure 04.05: Mammalian genes for DHFR have the same relative organization of rather short exons and very long introns.
4.5 Exon Sequences Under Negative Selection Are Conserved but Introns Vary • Comparisons of related genes in different species show that the sequences of the corresponding exons are usually conserved, but the sequences of the introns are much less similar. • Introns evolve much more rapidly than exons because of the lack of selective pressure to produce a polypeptide with a useful sequence.
Figure 04.06: The sequences of the mouse βmaj- and βmin-globin genes are closely related in coding regions. Data provided by Philip Leder, Harvard Medical School
4.6 Exon Sequences Under Positive Selection Vary but Introns Are Conserved • Under positive selection an individual with an advantageous survives (i.e., is able to produce more fertile progeny) relative to others without the mutation. • Due to intrinsic genomic pressures, such as that which conserves the potential to extrude stem-loops from duplex DNA, introns evolve more slowly than exons that are under positive selection pressure.
Figure 04.07: The sequences of snake venom phospholipase genes differ in coding regions, but are closely related in introns and flanking regions. Modified from D. R. Forsdyke, Conservation of Stem-Loop Potential in Introns of Snake Venom Phospholipase A2 Genes: An Application of FORS-D Analysis, Mol. Biol. Evol., vol. 12 (6), pp. 1157-1165, by permission of Oxford University Press.
4.7 Genes Show a Wide Distribution of Sizes Due Primarily to Intron Size and Number Variation • Most genes are uninterrupted in S. cerevisiae but are interrupted in multicellular eukaryotes. Figure 04.08: Most genes are uninterrupted in yeast, but most genes are interrupted in flies and mammals.
4.7 Genes Show a Wide Distribution of Sizes Due Primarily to Intron Size and Number Variation • Exons are usually short, typically encoding fewer than 100 amino acids. Figure 04.10: Exons encoding for polypeptides are usually short.
4.7 Genes Show a Wide Distribution of Sizes Due Primarily to Intron Size and Number Variation • Introns are short in unicellular/oligocellular eukaryotes but can be many kb in multicellular eukaryotes. • The overall length of a gene is determined largely by its introns. Figure 04.11: Introns range from very short to very long.
4.8 Some DNA Sequences Encode More Than One Polypeptide • The use of alternative translation initiation or termination codons allows multiple variants of a polypeptide chain. • overlapping gene – A gene in which part of the sequence is found within part of the sequence of another gene. Figure 04.12: Two proteins can be generated from a single gene by starting (or terminating) expression at different points.
4.8 Some DNA Sequences Encode More Than One Polypeptide • Different polypeptides can be produced from the same sequence of DNA when the mRNA is read in different reading frames (as two overlapping genes). Figure 04.13: Two genes may overlap by reading the same DNA sequence in different frames.
4.8 Some DNA Sequences Encode More Than One Polypeptide • Otherwise identical polypeptides, differing by the presence or absence of certain regions, can be generated by differential (alternative) splicing when certain exons are included or excluded. • This may take the form of including or excluding individual exons, or of choosing between alternative exons.
Figure 04.15: Alternative splicing uses the same pre-mRNA to generate mRNAs that have different combinations of exons.
4.9 Some Exons Can Be Equated with Protein Functional Domains • Proteins can consist of independent functional modules the boundaries of which, in some cases, can be equated with those of exons. Figure 04.16: Immunoglobulin light and heavy chains are encoded by genes whose structures correspond to the distinct domains in the protein.
4.9 Some Exons Can Be Equated with Protein Functional Domains • The exons of some genes appear homologous to the exons of others, suggesting a common exon ancestry. Figure 04.17: The LDL receptor gene consists of 18 exons. Triangles mark the positions of introns.
4.10 Members of a Gene Family Have a Common Organization • gene family – A set of genes within a genome that encodes related or identical proteins or RNAs. • The members were derived by duplication of an ancestral gene followed by accumulation of changes in sequence between the copies. • Most often the members are related but not identical.
4.10 Members of a Gene Family Have a Common Organization • superfamily – A set of genes all related by presumed descent from a common ancestor, but now showing considerable variation. • A set of homologous genes (homologs) should share common features that preceded their evolutionary separation. Figure 04.19: The rat insulin gene with one intron evolved by loss of an intron from an ancestor with two introns.
4.10 Members of a Gene Family Have a Common Organization • All globin genes have a common form of organization with three exons and two introns, suggesting that they descended from a single ancestral gene. Figure 04.18: The exon structure of globin genes corresponds to protein function, but leghemoglobin has an extra intron in the central domain.
4.10 Members of a Gene Family Have a Common Organization • Intron positions in the actin gene family are highly variable, which suggests that introns do not separate functional domains. Figure 04.20: Actin genes vary widely in their organization.
4.11 There Are Many Forms of Information in DNA • Genetic information includes not only that related to characters corresponding to the conventional phenotype, but also that related to characters (pressures) corresponding to the genome “phenotype.” • In certain contexts, the definition of the gene can be seen as reversed from “one gene-one protein” to “one protein-one gene.” • Positional information may be important in development.
4.11 There Are Many Forms of Information in DNA • Sequences transferred “horizontally” from other species to the germline could land in introns or intergenic DNA and thence transfer “vertically” through the generations. • Some of these may be involved in intracellular nonself-recognition.
