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Dr.Alia Shoeib Antibiotics and Plasmids Plant &Microbiology Dept. Micro.623 Definition Molecular biology is the study of biology at a molecular level. Molecular biology chiefly concerns itself with understanding the interactions between the various systems of a cell, including the interrelationship of DNA, RNA and protein synthesis and learning how these interactions are regulated. Molecular genetics is the field of biology which studies the structure and function of genes at a molecular level. Molecular genetics employs the methods of genetics and molecular biology. MOLECULAR GENETICS Genes ---> Enzymes ---> Metabolism Central Dogma of Molecular Biology* DNA -transcription--> RNA -translation--> Protein What is a GENE = ? DNA is the genetic material... - a discrete piece of deoxyribonucleic acid - linear polymer of repeating nucleotide monomers nucleotides* --> A adenine, C cytosine T thymidine, G guanine --> polynucleotide* INFORMATION PROCESSING & the CENTRAL DOGMA - the letters of the genetic alphabet... are the nucleotides A, T, G, & C Current dogma of Molecular Biology DNA --> RNA --> Proteins, (proteins supposedly regulate gene expression) figure* - unit of information is CODON = genetic 'word' 1 a triplet sequence of nucleotides CAT in a polynucleotide 3 nucleotides = 1 codon (word) = 1 amino acid - the definition of (codon) word = amino acid Size of Human Genome: ≈ 3,000,000,000 base pairs or 1.5b in single strand genes ≈ 500,000,000 possible codons (words or amino acids) 1. Transformation* Experiments of Fred Griffith... (1920's) Streptococcus pneumoniae - pathogenic S strain & benign R transforming 'principle' is genetic element 2. Oswald Avery, Colin MacLeod, & Maclyn McCarty... (1940's) suggest the transforming substance* is DNA molecules, but... Gene expression is the multi-step process by which a gene's information is converted into the structures and functions of a cell, following the central dogma of molecular biology. Recombinant DNA is DNA that has been created artificially. DNA from two or more sources is incorporated into a single recombinant molecule. A bacterial artificial chromosome (BAC) is a DNA construct, based on a fertility plasmid, used for transforming and cloning in bacteria, usually E. coli. Its usual insert size is 150 kbp, with a range from 100 to 300 kbp. BACs are often used to sequence the genetic code of organisms in genome projects, for example the Human Genome Project. A short piece of the organism's DNA is amplified as an insert in BACs, and then sequenced. Finally, the sequenced parts are rearranged in silico, resulting in the genomic sequence of the organism. 2 To be useful, the recombinant molecule must be replicated many times to provide material for analysis, sequencing, etc. Producing many identical copies of the same recombinant molecule is called cloning. Cloning can be done in vitro, by a process called the polymerase chain reaction (PCR). Here, however, we shall examine how cloning is done in vivo. Cloning in vivo can be done in unicellular prokaryotes like E. coli unicellular eukaryotes like yeast and in mammalian cells grown in tissue culture. In every case, the recombinant DNA must be taken up by the cell in a form in which it can be replicated and expressed. This is achieved by incorporating the DNA in a vector. an example of cloning using E. coli as the host and a plasmid as the vector. vector Plasmids are sometimes called "vectors", because they can take DNA from one organism to the next. Not all vectors are plasmids, however. We commonly use engineered viruses, for example bacteriophage lambda, which can carry large pieces of foreign DNA. Plasmids In addition to the nucleoid, many bacteria often contain small nonchromosomal DNA molecules called plasmids. Plasmids usually contain between 5 and 100 genes. Plasmids are not essential for normal bacterial growth and bacteria may lose or gain them without harm Transposons (transposable elements or "jumping genes") are small pieces of DNA that encode enzymes that transpose the transposon, that is, move it from one DNA location to another. Transposons may be found as part of a bacterium's nucleoid (conjugative transposons) or in plasmids and are usually between one and twelve genes long. A transposon contains a number of genes, coding for antibiotic resistance or other traits, flanked at both ends by insertion sequences coding for an enzyme called transpoase. Transpoase is the enzyme that catalyzes the cutting and resealing of the DNA during 3 transposition. Thus, such transposons are able to cut themselves out of a bacterial nucleoid or a plasmid and insert themselves into another nucleoid or plasmid and contribute in the transmission of antibiotic resistance among a population of bacteria. TRANSPOSONS - pieces of DNA prone to moving & creating repeat sequences LINE - long interspersed nuclear element holds promoter & 2 genes: RT & integrase Plasmids can also acquire a number of different antibiotic resistance genes by means of integrons. Integrons are transposons that can carry multiple gene clusters called gene cassettes that move as a unit from one piece of DNA to another. An enzyme called integrase enables these gene cassettes to integrate and accumulate within the integron. In this way, a number of different antibiotic resistance genes can be transferred as a unit from one bacterium to another. Biotechnology Restriction Enzymes : definition: enzymes that cut DNA in specific places function: inactivate foreign DNA which can derange metabolism breaks only palindrom sequences, i.e. those exhibiting two-fold symmetry examples Important in DNA research, i.e. sequencing, hybridization Companies purify and market restriction enzymes Modification Enzymes protect destruction of native DNA by native restriction enzymes mechanism A nucleotide can be a substrate for a particular restriction enzyme or modification enzyme but not both ===================================================== 4 Gene Amplification: polymerase chain reaction (PCR) to make multiple copies of a specific gene ====================================================== Dr.Alia Shoeib Plant &Microbiology Dept. Antibiotics and Plasmids Micro.623 GENE Expression the Central Dogma of Molecular Biology depicts flow of genetic information Transcription - copying of DNA sequence into RNA Translation - copying of RNA sequence into protein DNA sequence -------> RNA sequence -----> amino acid sequence TAC AUG MET triplet sequence in DNA --> codon in mRNA ----> amino acid in protein Information : triplet sequence in DNA is the genetic word [codon] GENETIC CODE... ...is the sequence of nucleotides in DNA, but shown as a mRNA code* ...specifies sequence of amino acids to be linked into the protein GENETIC CHANGE - a change in DNA nucleotide sequence - 2 significant ways mutation & recombination 1. MUTATION - a change in an organism's DNA*that results in a different codon = different amino acid sequence Point mutation - a single to few nucleotides change - deletions, insertions, frame-shift mutations* [CAT] 2. Recombination (Recombinant DNA) newly combined DNA's that [glossary]* 5 can change genotype by insertion of NEW (foreign) DNA molecules into recipient cell 3. transformation* - absorption of 'foreign' DNA by recipient cells 4. BACTERIAL CONJUGATION* - involves DNA plasmidsg* (F+ & R = resistance) conjugation is primitive sex-like reproduction in bacteria [Hfr*] 5. VIRAL TRANSDUCTION - via a viral vector ( lysogeny & TRANSDUCTION* ) general transduction - pieces of bacterial DNA are packaged w viral DNA during viral replication restricted transduction - a temperate phage goes lytic carrying adjacent bacterial DNA into virus particle 6. DESIGNER GENES - man-made recombinant DNA molecules Designer Genes - Genetic Engineering - Biotechnology 6