Chapter 5 Exploring Genes and Genomes

What is a Gene? A gene is the molecular unit of heredity of a living organism DNA (Protein) DNA (Regulatory) RNA (Regulatory) Genome ?

Recombinant DNA

Recombinant DNA Techniques What does it mean “to clone” (or cloning) a gene? What are restriction enzymes? What is a cDNA library and reverse transcription? Expression vectors. Transgenic mice. What is the polymerase chain reaction (PCR)? How is RNA interference used to reveal the function of genes? DNA microarrays Human Genome Project

Cloning a Gene 1. Clone: a collection of molecules or cells, all identical to the original Cloning a gene is to make many copies of it Gene can be an exact copy or an altered version of a natural gene 2. Plasmids Plamids are naturally occurring extrachromosomal DNA (circular dsDNA) Plasmids can be cleaved by restriction enzymes These recombinant molecules can be replicated and propagated 3. Cloning Vectors: plasmids that can be modified to carry new genes, must have: Origin of replication A selectable marker (antibiotic resistance gene) A cloning site (site where insertion of foreign DNA will not disrupt replication or inactivate essential markers)

3.7 Bacterial Plasmid Genetic Complementation AmpR AmpS AmpR = ampicillin resistant & AmpS = ampicillin sensitive Genetic Complementation

Plasmids as Cloning Vectors One of the first widely used cloning vectors was the plasmid pBR322. Note the antibiotic resistance genes (ampr and tetr).

Any DNA Sequence Can Be Cloned Nuclease cleavage at a restriction site linearizes the circular plasmid so that a foreign DNA fragment can be inserted. Recombinant plasmids are hybrid DNA molecules consisting of plasmid DNA sequences plus inserted DNA elements (pink). Such hybrid molecules are called chimeric plasmids. An EcoRI restriction fragment of foreign DNA can be inserted into a plasmid.

Bacterial Conjugation William Hayes (1953) determined that genetic transfer occurred in one direction. Therefore, the transfer of genetic material in E. coli is not reciprocal. One cell acts as donor, and the other cell acts as the recipient. In bacterial gene transfer (conjugation), one organism receives genetic information from a donor; the recipient is changed by that information. During bacterial conjugation, the pilus brings two bacteria together, allowing a bridge to form between the two cells. Then one strand of plasmid DNA passes into the recipient bacterium, and each single strand becomes double stranded again.

Transduction

(Anne, I vote more cars race Rome to Vienna) Restriction Enzymes 1. Restriction endonucleases are bacterial enzymes used to cut DNA. DNA ligase catalyzes the joining of DNA fragments. Restriction enzymes and DNA ligase are used to cut DNA into fragments and then splice them together in new combinations. 2. Recombinant DNA is a DNA molecule made in the laboratory that is derived from at least two genetic sources. Restriction enzymes recognize palindromic DNA sequences: (Anne, I vote more cars race Rome to Vienna) 5′…….GAATTC……3′ 3′…….CTTAAG……5′ 4. Some make straight cuts, others make staggered cuts, resulting in overhangs or “sticky ends”. Sticky ends can bind by base pairing to other sticky ends. 5. Fragments from different sources can be joined. Then ligase catalyzes formation of covalent bonds between adjacent nucleotides at fragment ends, joining them to form a single, larger molecule.

Restriction Enzymes

Restriction Enzymes Use of restriction enzymes to generate recombinant DNA. The vector DNA and the target DNA are cleaved by restriction endonucleases to generate ends that can be joined together. In cases where sticky ends are produced, the two molecules join by annealing (base pairing) of the complementary ends. The molecules are then covalently attached to one another in a reaction catalyzed by DNA ligase.

Restriction Enzymes

Selection To determine which of the host cells contain the new sequence, the recombinant DNA includes selectable marker genes, such as genes that confer resistance to antibiotics.

DNA Library A DNA library is a collection of DNA fragments that comprise the genome of an organism. The DNA is cut into fragments by restriction enzymes, and each fragment is inserted into a vector, which is used to produce a colony of recombinant cells.

cDNA Libraries cDNAs are DNAs copied from mRNA templates. cDNA libraries are constructed by synthesizing cDNA from purified cellular mRNA. Because most eukaryotic mRNAs carry 3'-poly(A) tails, mRNA can be selectively isolated from preparations of total cellular RNA by oligo(dT)-cellulose chromatography DNA copies of the purified mRNAs are synthesized by first annealing short oligo(dT) chains to the poly(A) tails. These serve as primers for reverse transcriptase-driven synthesis of DNA

cDNA are Prepared from mRNA Reverse transcriptase is an enzyme that synthesizes a DNA strand, copying RNA as the template DNA polymerase is then used to copy the DNA strand and form a double- stranded duplex DNA Linkers are then added to the DNA duplexes rendered from the mRNA templates The cDNA is then cloned into a suitable vector Once a cDNA derived from a particular gene has been identified, the cDNA becomes an effective probe for screening genomic libraries for isolation of the gene itself

Reverse Transcription

Recombinant Protein

Expression Vectors Expression vectors are engineered so that the RNA or protein products of cloned genes can be expressed. Expression vectors carrying the promoter recognized by the RNA polymerase of bacteriophage SP6 are useful for the production of multiple RNA copies of any DNA inserted at the polylinker.

Expression Vectors To express a eukaryotic protein in E. coli, the eukaryotic cDNA must be cloned in an expression vector that contains regulatory signals for transcription and translation.

Expression Vectors Some expression vectors carry cDNA inserts cloned directly into the coding sequence of a protein-coding gene.

Transgenic Mouse

Transgenic Mouse

Polymerase Chain Reaction A small sample of DNA serves as template for DNA polymerase Make complementary primers Add primers in more than 1000-fold excess Heat to make ssDNA, then cool Run DNA polymerase (usually Taq) Repeat heating, cooling, polymerase cycle

In Vitro Mutagenesis PCR-based site-directed mutagenesis Template DNA strands are separate and amplified by PCR. Following many cycles of PCR, the DNA product can be used to transform E. coli cells. The plasmid DNA can be isolated and screened for the presence of the unique restriction site (by restriction enzymes).

RNA Interference (RNAi) a biological process in which RNA molecules inhibit gene expression or translation, by neutralizing targeted mRNA molecules siRNA miRNA

siRNA vs miRNA

siRNA vs miRNA https://www.youtube.com/watch?v=cK-OGB1_ELE

RNA Interference (RNAi) RNA interference (RNAi) has emerged as a method of choice in eukaryotic gene inactivation RNAi leads to targeted destruction of a selected gene’s transcript The consequences following loss of gene function reveal the role of the gene product in cell metabolism

DNA Microarray DNA microarray technology provides a large array of sequences for hybridization experiments. A series of DNA sequences are attached to a glass slide in a precise order. The slide has microscopic wells which each contain thousands of copies of sequences up to 20 nucleotides long.

DNA Microarray DNA microarrays can be used to identify specific single nucleotide polymorphisms or other mutations. If mRNA is to be analyzed, it is usually incubated with reverse transcriptase to make cDNA. Fluorescent dyes tag on the cDNAs and they are used to probe the DNA on the microarray. DNA chip technology has been developed to identify gene expression patterns in women with a propensity for breast cancer tumors to recur, a gene expression signature.

DNA Microarray

Human Genome Project National Institutes of Health (Francis Collins) International consortium Universities and research centers $3,000,000,000 ($3 billion) 1990 to 2003 “Chromosome walking” Celera Genomics (Craig Venter) Private company $300,000,000 ($300 million) 1998 to 2003 “Shotgun sequencing” Francis Collins Craig Venter

Human Genome Project 3.2 billion base pairs 25,000 genes produce 400,000 different proteins Removing different combinations of introns makes different proteins Only about 1.5% of genome encodes protein 98.5% encodes regulatory sequences, pseudogenes, and transposons