Through a study of genetics, we can understand the underlying basis for how microorganisms can do what they do (metabolism), and ultimately all life processes.

OVERVIEW OF GENETIC PROCESSES
The Basis of Heredity

• All the information of cells is stored in their genetic material; DNA. Certain viruses alternatively use RNA for information storage; all other organisms use double-stranded DNA. The transmission of genetic information from an organism to its progeny is called heredity.

• The entire complement of genetic material of an organism is called its genome. In most prokaryotes, the genome consists of one large molecule of DNA (usually circular), called a chromosome, in eukaryotes, there are several chromosomes (always linear).

• The basic unit of heredity is called a gene, consisting of a linear sequence of nucleotides that form a functional unit; for example, one gene codes for one polypeptide.
• Remember, DNA is made of repeating units called nucleotides
• Nucleotides consists of 3 components: nitrogenous base (A,T,C,G), deoxyribose (sugar), and a phosphate group.

• The genotype of an organism is its genetic makeup (all particular characteristics); a phenotype is an observable characteristic.
• Prokaryotes by and large have haploid genomes (i.e. one copy of each gene, located on the single chromosome); while eukaryotes have diploid genomes (i.e. there are two copies of each gene which are located on pairs of chromosomes). In the case of eukaryotes, there may be different information at the same location, or locus on one or the other of the pair of chromosomes. The genes with different information at the same locus are called alleles; for example, the A, B, O alleles associated with human blood types.

• A change in a gene, which is passed on to the cell's progeny, is called a mutation. This change may or may not give rise to a change in phenotype.

II. Nucleic Acids in Information Storage and Transfer
Information Storage:

• All genomic DNA is composed of paired strands of linear sequences of the four nucleotides, A, C, G, and T.  Typical units of the genome, or genes in prokaryotes, are about 1000 base pairs in length.
• Genetic code: determines how a nucleotide sequence is converted into amino acids of a protein

• There are two information transfer processes involving DNA; (1) replication, which is duplication of itself, and (2) transcription, which is the transcribing of the DNA sequence to another nucleic acid, RNA.
• One class of RNA transcript, called messenger RNA, is used for translation, converting the sequence of nucleotides to a sequence of amino acids in a polypeptide.

III. REPLICATION OF DNA

• E. coli- chromosome is about 4 million base pairs and about 1mm long (1000 times longer than the entire cell!)
• The two DNA strands are held together by hydrogen bonds between complementary bases; adenine and thymine, and cytosine and guanine.
• There is directionality of the DNA strands conferred by the sugar-phosphate backbone, and the relative directionality of the pairs of strands are opposite to one another, or anti-parallel.
• Remember: replication of DNA is semi-conservative
• Replication of the genomic DNA in cells begins at specific points, usually proceeding in both directions at the same time, or bidirectionally. The DNA has to unwind and the two strands separate in order for new DNA to be synthesized, and so the formation creates a replication fork structure. Each "parent strand" serves as the pattern or template for the synthesis of the new DNA strand. When completed, the new pair of DNA strands consists of one parent strand and one newly synthesized strand. This replication mechanism is called semi-conservative because one parent strand is always conserved.
• All cells have enzymes that can only add new nucleotides to the 3' end of a nucleic acid; either RNA or DNA. This means the cells need to use two different mechanisms of replication, depending on which strand is being replicated.
• The leading strand is synthesized in a continuous fashion 5' to 3'. Remember, always towards the replication fork!!!
• The lagging strand is synthesized in a discontinuous fashion 5' to 3', giving rise to a series of short DNA fragments that are then joined together. Remember, always away from the replication fork

Special Aspects of DNA Replication

• Enzymes that replicate DNA are called DNA polymerases; besides functioning unidirectionally, no DNA polymerase can begin a nucleic acid chain. So, regardless of which strand, replication begins with the synthesis of a small "starter" nucleic acid by a special enzyme, called primase. The starter nucleic acid, which consists of RNA, is called a primer.
• The linear chromosomes present in all eukaryotes require a special mechanism to complete the replication of the ends of the chromosomes; otherwise they would become shorter each time they were replicated. This mechanism uses an enzyme called telomerase that attaches to the linear end, and has a "built-in" primer.

IV. PROTEIN SYNTHESIS
Transcription

• For life processes, there is constant synthesis of proteins. Protein synthesis begins with the transcription of the information contained in genes (DNA) into RNA. This begins with unpairing of a small stretch of DNA that exposes a single-stranded region to serve as the template for the synthesis of the RNA. In RNA-DNA pairing, the ribonucleotide uracil pairs with the deoxyribonucleotide adenine.
• Transcription always occurs 5' to 3', and requires energy in the form of high energy phosphate bonds. The enzyme that catalyzes transcription is called RNA polymerase.
• In eukaryotes, transcription takes place in the nucleus (as does DNA replication). Also, the RNA that encodes polypeptides contains stretches that will not be used, called introns, and are first removed before protein synthesis occurs. The stretches that are used are called exons. The process that joins the exons together by removing the introns is called splicing.

Kinds of RNA
There are three kinds of RNAs within cells; ribosomal RNA, messenger RNA, and transfer RNA. All are synthesized by transcription of DNA sequences.

• Ribosomal RNAs (rRNAs) are an integral part of the protein-synthesizing machinery of the cell, called the ribosome which in addition to RNA contains several polypeptides.
• Messenger RNAs (mRNAs) are those RNA molecules that direct which sequence of amino acids are synthesized on the ribosomes. The process of protein synthesis on ribosomes is called translation.
• The information that identifies which amino acid should be incorporated at each position is contained in the nucleotide sequence of the mRNA in groups of three nucleotides each, called codons.
• The first codon of the mRNA is called a "start" codon (AUG), and it always codes for the amino acid methionine; the last codon is called a "terminator" codon that tells the ribosome to end synthesis.
• There are 61 sense codons and 3 nonsense codons (UAG, UGA, UAA)
• Which codon specifies which amino acid is called the genetic code; those codons that do not code for amino acids are called nonsense codons, and correspond to the terminator codons.
• The genetic code is degenerate - a particular amino acid can be coded by more than one codon.
• Transfer RNAs (tRNAs) are these RNA molecules that carry amino acids to the ribosome where they are joined to previously added amino acids (starting with methionine). The tRNAs are 75 to 80 nucleotides long, and have regions with specific functions.
• One region contains the anticodon, which is a sequence of three residues complementary to a codon present on the mRNA.
• The ribosome has 2 sites: the A (acceptor site) and the P (peptide) site

Translation

• Protein synthesis uses 80 to 90% of a bacterial cell's energy. The amount of messenger RNA synthesized determines how much protein gets made.
• Translation occurs by (1) attachment of ribosomes to mRNA, (2) tRNAs bring amino acids to the ribosomes (3) the amino acid is transferred from the tRNA to the end of the growing polypeptide chain (4) the tRNA dissociates from the ribosome.
• Specification of which amino is added occurs through matching of the anticodon on the tRNA with the codon of the mRNA.

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Types of Mutations and Their Effects
All changes in DNA that are passed on to progeny are called mutations. The genetic information of an organism is called its genotype, so a mutation will alter the organism's genotype.

Two classes of mutations involving only one or two nucleotides are point mutations and frameshift mutations.

I. A point mutation is the replacement of a single nucleotide by another. If this occurs within the
    coding sequences for a polypeptide, there are three possible outcomes:
    a) -The new sequence codes for a different amino acid; this is called a missense mutation.
    b) -The new sequence codes for the same amino acid as the original sequence; this is called a
        silent mutation.
    c) -The new sequence becomes a nonsense codon; this is called a nonsense mutation.

II. A frameshift mutation is a mutation in which there is either a deletion or an insertion of one
    or two nucleotides within the coding sequences for a polypeptide. This is called a frameshift,
    because the normal register for the codons (the three bases that code for an amino acid) becomes
    shifted. There can also be large deletions and insertions, as well, which may or may not shift the
    register for the codons.

Phenotypic Variation
If a mutation, a change in an organism's genotype, leads to an observable difference, then the mutation has also altered the phenotype of the organism.

I. Nutritional requirements: The mutant is with the deficiency is called an auxotroph; the
    non-mutant is called a prototroph. By determining what the new nutritional requirement is, genes
    coding for various metabolic enzymes can be identified

II. Temperature sensitivity:  This often involves a mutation such that the mutant polypeptide may
    be unimpaired at one temperature, but rendered non-functional at another temperature (for
    example, it becomes more easily denatured when the temperature is elevated).

Spontaneous versus Induced Mutations

• Spontaneous mutations are those that occur in the absence of any agent known to cause changes in DNA. They arise during replication of DNA through errors in the base pairing of nucleotides in the old and new strands of DNA.
• Typical rates for spontaneous mutations are 1 in 1 million in Bacteria.
• Induced mutations are those that are induced by agents called mutagens that include both chemical agents and radiation.

• A base analog is a compound that is similar in structure to a natural base, and can become misincorporated during DNA synthesis. When the DNA is then replicated, the structure is such that an incorrect nucleotide is incorporated into the new strand.
• Those agents that add alkyl groups, such as a methyl group to a nucleotide are called alkylating agents. Again, the added group causes mispairing when the DNA is replicated.
• Those agents that remove an amino group from a base are called deaminating agents. This causes adenine to resemble guanine so that it is paired with cytosine rather than thymine when DNA synthesis occurs.
• Acridine derivatives cause frameshift mutations because it can become inserted into the DNA double helix, distorting the helix and allowing the addition or deletion of one or more base pairs.

• Ultraviolet radiation causes the formation of pyrimidine dimers, so that when replicated it is more common for the wrong base to be inserted.
• Ionizing radiation: (X-rays and gamma rays) breaks chemical bonds in molecules and can generate free radicals that are highly reactive and can attack DNA.

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To isolate and identify mutants, there are two general methods;

-Selection can be used when the mutation confers a new metabolic ability or property that allows the mutant to grow under conditions that do not allow the non-mutant to grow.

-If a mutation leads to a loss of a metabolic capability, such as auxotrophy, replica plating can be used.

The Ames Test
Because human cancers can be induced by substances that alter DNA, it is useful to have a test for potentially mutagenic agents. Bacteria can be used to screen for such agents, in the Ames test. The Bacteria are auxotrophic for histidine, but can undergo another mutation that now allows them to synthesize histidine. If an agent is mutagenic, it will increase the rate at which the Bacteria develop the ability to synthesize histidine.

It is essential to understand that a negative test does not rule out that it may affect humans; nor does a positive test always mean that the agent is mutagenic for humans.