SWARTZ MICROBIOLOGY NOTES 57 Chapter 9 MICROBIAL GENETICS INTRODUCTION TO GENETICS AND GENES: UNLOCKING THE SECRETS OF HEREDITY Genetics is the study of the inheritance or heredity of living things. Organismic genetics studies the heredity of the whole organism; chromosomal genetics studies the characteristics and actions of chromosomes, and molecular genetics studies the biochemistry of the genes. THE NATURE OF THE GENETIC MATERIAL The Levels of Structure and Function of the Genome: A genome is a complete set of genetic material (genes) in a cell. For example, an ovum or a sperm contains a genome. The genetic material of living things is predominantly DNA. However, the genetic material of some viruses such as HIV and Ebola is RNA. A gene can be defined as a sequence of DNA which codes for the synthesis of one polypeptide. Bacterial chromosome consists of a single molecule of double-stranded deoxyribonucleic acid (DNA) in ring shape which is in association with histonelike proteins. A bacterium contains one chromosome. It contains hereditary information which is passed from one generation to the next generation. The procaryotic chromosome is not surrounded by a nuclear membrane. A bacterium may contain one or more extra piece of chromosomes called plasmids. Plasmids are circular, double-stranded DNA. Linear plasmids have also been found in a few species of bacteria. Plasmids may contain genes responsible for antibiotic resistance. They have been used as vectors to transfer the foreign genes into the bacterial cells in genetic engineering techniques. The Size and Packaging of Genomes: The smallest viruses contain 4 or 5 genes. Escherichia coli contains about 4,000 genes and human cell contains about 100,000 genes. The DNA of E. coli if unwound and stretched out linearly, it measures about 1 mm. If all the DNA in 46 chromosomes in a human cell are linked together to form a linear DNA, it will measure about 6 feet. If all the DNAs in a human body (50 trillion cells) are linked together, its length is more than enough to cover a distance to and back from the moon. A book with one million pages will be made if all the bases (A, T, C, G) found in 46 chromosomes in a human cell are printed. THE DNA CODE: A SIMPLE YET PROFOUND MESSAGE DNA consists of building blocks called nucleotides. A nucleotide consists of a phosphate, a sugar (deoxyribose) and a nitrogenous base (adenine, guanine, cytosine and thymine). Nucleotides link together to form a polynucleotide (strand).. A DNA consists of two strands arranged in an anti-paralleled direction. If one strand goes from the 3' to 5' direction, the other strand always goes in the 5' to 3' direction. These two strands are held together by hydrogen bonds between their nitrogenous bases according to the base-pairing rules: adenine with thymine, and cytosine with guanine. The number of hydrogen bonds between adenine and thymine is 2, and that between cytosine and guanine is 3. THE SIGNIFICANCE OF DNA STRUCTURE The nitrogenous bases influence DNA in two major ways: SWARTZ MICROBIOLOGY NOTES 58 1. Maintenance of the code during reproduction: During cell division, the DNA strands separate, each serves as a template for the synthesis of a new strand. In this manner, each daughter cell will inherit the identical DNA. 2. Providing variety: The order of bases along the DNA strands is unique in each organism. A gene is a specific sequence of bases. An endless number of sequences can be made from four nitrogenous bases. DNA REPLICATION: PRESERVING THE CODE AND PASSING IT ON DNA replication is the process in which DNA is duplicated. In E. coli, the process takes about 20 minutes. The Overall Replication Process: The process involves the following basic steps: (1) uncoiling of DNA, (2) unzipping of the hydrogen bonds so each strand serves as a template for the synthesis of a new strand, and (3) synthesis of a new strand whose bases are complementary to those of the old strand. DNA synthesis is described as semi-conservative type because each old strand is used as a template for the synthesis of a new strand. So, in every DNA molecule one strand is always an old one and another strand is always a new one. Refinements and Details of Replication: The circular bacterial DNA replicates by a special configuration called a replicon (Fig. 9.7, p. 264). During bacterial DNA replication, the circular DNA double helix unwinds due to the action of the enzyme DNA gyrase at a specific site. Replicons are found only in prokaryotic DNA, but not in eukaryotic DNA. The hydrogen bonds are broken by the enzyme DNA helicase ('unzipping enzyme"). (Some books also describe that DNA helicase can also unwind DNA.) The helix separates, forming two replication forks (Fig. 9.7 & 9.8, p. 264-265). The forks move in opposite directions around the circular DNA until they meet. As the DNA strands separate, each strand is used as a template to synthesize a new strand. Synthesis of new DNA strands can occur in only one direction, from the 5' phosphate end to the 3' hydroxyl end of DNA. That is, the nucleotides have to be added from the old 3' hydroxyl end to the 5' phosphate end of DNA. Two new strands are assembled in an anti-parallel direction. The assembly of nucleotides on the old 3'-5' strand is straight forward. It is accomplished by DNA polymerase III. On the old 5'-3' strand, the assembly is discontinuous, forming segments of new nucleotides. Each segment always starts with a RNA primer of about 10 bases. It is synthesized by RNA polymerase. A primer provides a point to which true DNA nucleotides may be added by DNA polymerase III. After a few DNA nucleotides have been connected to the RNA primer by DNA polymerase III, the primer is erased (degraded) by DNA polymerase I (eraser enzyme). The bases which have been erased are replaced with true DNA nucleotides by DNA polymerase II (repair enzyme). (Some books also describe that DNA polymerase I can also remove RNA primer and repair it. This book also says that DNA polymerase III can remove the RNA primers.) DNA fragments, called Okazaki fragments which consist of about 1,000 to 2,000 bases, are formed along the old 5'-3' strand. Many Okazaki fragments are formed in this manner. Each fragment always starts with a RNA primer. These Okazaki fragments are then joined together by DNA ligase (also called polynucleotide ligase or joining enzyme) to form a new polynucleotide. This seemingly wasteful process may serve as a fail-safe procedure of DNA copying, since most errors occur at the beginning of the old copy where the 3' end has not yet been exposed. Elongation and Termination of the Daughter Molecules: The replicon looks like a half-open eye as one duplicating strand droops down (theta 0) (Fig. 9.8, p. 260). When two replication forks come full circle and meet, two DNA molecules are produced. SWARTZ MICROBIOLOGY NOTES 59 The rate of synthesis is about 500 (750 in some bacteria) nucleotides per second in procaryotes and about 50 nucleotides per second in eucaryotes. Other Variations on the Theme: DNA replication in eucaryotes starts at many sites along the DNA. Each site forms a replication bubble. Hundreds of these bubbles eventually fuse when two linear double-stranded DNA molecules are formed. A type of replication called rolling circle occurs in bacteriophages and plasmids (Fig. 9.9, p 265). A new strand is made from the parent DNA as it rotates. A complementary strand is then made from the new strand to form a double-stranded circular DNA. DNA replication is precise. However, occasionally mistakes are made once in about 100,000 to 1 million bases. They are repaired by DNA polymerase III. If mistakes are not repaired, the cell will undergo mutation. APPLICATIONS OF THE DNA CODE: TRANSCRIPTION AND TRANSLATION The central dogma (theme) of molecular biology is the concept that genetic information flows from DNA to RNA to protein. The synthesis of RNA from DNA is called transcription, and the synthesis of protein from RNA is called translation. Exceptions to this concept are found in some RNA viruses which convert RNA to other RNA, and in retroviruses which convert RNA to DNA. Transcription in eukaryotic cells occurs in the nucleus, while translation occurs on ribosomes. THE GENE-PROTEIN CONNECTION There are three basic categories of genes: structural genes code for proteins, genes that code for RNA, and regulatory genes control gene expression. The genotype is the genetic makeup of an organism, and the phenotype is the observable characteristics of an organism (i.e. shape of cell). The Triplet Code and the Relationship to Proteins: The three adjacent bases on one DNA strand constitute a triplet. When a DNA sequence is transcribed into a sequence of RNA, each triplet is called a codon. A codon codes for one amino acid. The uniqueness of an organism is due to the uniqueness of its DNA sequence which dictates the specific order of amino acids in a polypeptide. The relationship between DNA and protein functions are: (1) the primary structure of a protein determines its characteristic shape and function, (2) proteins determine phenotype, and (3) DNA is a blueprint of life. It tells a cell what proteins to make. THE MAJOR PARTICIPANTS IN TRANSCRIPTION AND TRANSLATION RNAs: Tools in the Cell's Assembly Line RNA is a single-stranded molecule (it may also form a hairpin loop, a secondary structure, or a tertiary structure). RNA contains uracil (in place of thymine found in DNA) and ribose (in place of deoxyribose found in DNA).