Microbiology Notes Exam 2 (Lectures 9&10) – Flashcards

DNA

• How? Prof. McCleary didn't go in depth in this lecture but she did when she spoke of protein synthesis some weeks back.
• DNA is a nucleic acid with a phosphate deoxyribose backbone and four nucleotide bases which appear in varying sequence.
• The sequence of these bases is read like a langauge by ribsomes. 
• To ribosomes, every three bases (e.g. ATG) is a one word instruction calling for a complementary set of bases (e.g. TAC) and an attendent specific amino acid. 
• By way of these instructions and the actions of ribosomes and 3 types of RNA amino acids are strung together to form polypeptide chains. 
• These chains form the the functional (ENZYMES) and structural proteins which build and make the body function. 

Deoxyribose

• The image below shows deoxyribose. It also indicated the position of both the thre prime and five prime carbons. 

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• This image shows the physcial relationship between deoxyribsoe and the phosphate and nucleotide base in a DNA molecule

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Adenine (A) Guanine (G) Thymine (T) and Cytosine (C)

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complementary base pairing

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• What pairs with each of the following?
• Cytosine pairs with Guanine
• Thymine pairs with Adenine
• Adenine pairs with Thymine
• Guanine pairs with Cytosine

• A - T 
• G - C
• T - A
• T - A
• A - T
• C - G

A gene!

• Our genome is composed of> chromosomes (23 pairs or 46 chromosomes in humans, 1 in bacteria) > chromosomes are composed of genes > which are segments of DNA 

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A gene is a segment of the DNA that codes for specific producte

e.g. the gene for eye color, or for a flagella

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The organism's genome

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The single bacterial chromosome and its plasmid(s), if present. 

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• When people speak of the human genome they are usually referencing chromosomal DNA only. 
• Mitochondrial DNA is often not considered, though technically it is part of our genome. It allows mitochondria to replicate independently

Mitochondrial DNA

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Mitochondria

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• Mitochondrial DNA is inherited from one's mother

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• mitochondria are structures within eukaryotic cells that convert chemical energy from food into a form that cells can use, adenosine triphosphate (ATP).
• mitochondria are often called the "powerhouses of the cell" because of their central role in energy production. 
• NOTE: while bacteria produce energy via the electrong transport chain in their cell membrane, eukaryotic organisms produce it in the inner mitochondrial membrane or cristae. 

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• MORE EXERCISE > MORE MITOCHONDRIAL REPLICATION > MORE ENERGY FOR EXCERCISE 
• WHY?:
• Mitochondria are small structures found in most cells.  They are often referred as the "powerhouse" of the cell because they transform energy from food into cellular energy (ATP). 
• Because mitochondria have their own DNA they can replicate within the cell independently. 
• Excericise increases the replication of mitochondria in muscle cells "being worked", improving the body's ability to produce energy. 
• In other words, the more mitochondria you have, the more energy you can generate during exercise, the faster and longer you can exercise.

The organism's genome. 

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• The definitions for these words vary considerably from source to source, and even within single sources. 
• Prof. McClearly said the difference was semantic: different langauge used to say essentially the same thing. This implies we don't have to know the difference but it's probably helpful to know it, if only for reading. 
• Genotype is often used to talk about an individual's inherited genes. 
• Genome is often used to speak more broadly about the genetic material of a species. 

• Below are the definitions from the Human Genome Project's website:
• A genotype is an individual's collection of genes. 
• The term also can refer to the two alleles inherited for a particular gene. The genotype is expressed when the information encoded in the genes' DNA is used to make protein and RNA molecules. The expression of the genotype contributes to the individual's observable traits, called the phenotype.
• The genome is the entire set of genetic instructions found in a cell
• In humans, the genome consists of 23 pairs of chromosomes, found in the nucleus, as well as a small chromosome found in the cells' mitochondria. These chromosomes, taken together, contain approximately 3.1 billion bases of DNA sequence.

PHENOTYPE

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alleles

In this example, the alleles are for a flower's color. 

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• Knowing that type A is a codominant blood type with type B (if either is present, it will be expressed, always) we can say that the genotype can not have type B in it. 
• If it did, the B be expressed, resulting in type AB blood.
• Knowing that O has no markers, we can not say if it is present or absent.
• The genotype is either AA or AO.

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• These parents have a 1 in 4 chance of having a blue eyed child each time they reproduce.
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• Eukaryotic celdiploidls are diploid, meaning they have two genes that  code for each trait.

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• A haploid cell. 
• Haploid cells are in prokaryotic organisms.  
• They NOT EXCLUSIVE TO PROKARYOTES
• Gametes, fungi, some plants and some insects are also haploid. 
• Prokaryotic organisms only contain haploid cells. 

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HAPLOID, meaning they have only one copy of their genetic information.

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• Bacteria are haploid, therefore, given the right environment, their genes will be expressed.
• This is not true with diploid organisms. There is no way, for example, to make a person with a brown and blue allele for eye color, express the gene that they received for blue eyes. The dominant gene is always expressed. 

• Research using bacteria informs our understanding of how genetics works. 
• This is because bacteria are haploid, making it easier to tract the passage and expression of genetic traits among them. 
• It is "easier" due to the fact that one does not have to deal with dominance, codominance, blending, recessive traits etc.  

• No, because prokaryotic DNA is haploid. Only one gene controls for each expressed "trait". 
• * Allele: two genes that control for the same trait.

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• This was a little unclear. I'm going to ask. 
• I believe it is the process of replication, whereby a DNA molecule is unzipped and complimentarity of bases allows production of two identical daughter molecules.  
• It could also be the editting process, by which mismatched bases are removed. 

• Complementarity of bases allows DNA to be copied. 
• HOW?
• The two strands of DNA are complementary to one another.
• The base adenine will only pair with the base thymine and the base guanine will only pair with the base cytosine.
• Because of this, when the helix unwinds and replication occurs, each strand generates a copy of its original partner, resulting in 2 strands of identical DNA.

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Repeating deoxyribose sugar groups connected by phosphate groups.

• It is often called a sugar phosphate backbone, though Prof. McCleary sometimes simply calls it the "deoxyribose backbone". 

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It increases in size.

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Replication of DNA

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• That circle showed the entirety of a eukaryotic cell's life cycle. 
• The section with the letters PMAT represented the phases of mitosis. These are  P- prophase M- metaphase A- anaphase T- telophase.
• It wasn't clear to me if we had to know these
• The purpose of this drawing was to show us that DNA replication happened prior to mitosis, in interphase

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DNA replication occurs prior to mitosis. 

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• A DNA proofreading process.
• Prof. McCleary indicated this but other sources indicate that the proofreading happens concurrently with the replication, that is, as each section is laid down.
• I'll ask.  

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Accurate matching of base pairs in the DNA sequence

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• The cell has enzymes which allow the erroneously placed piece of DNA to be cut out and a new correct base to be spliced in. 

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• Because the DNA goes through a proofreading process before the divisional stage occurs. This proces corrects the errors. 

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A mutation

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All future generations of that cell. 

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Replication Fork (D)

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• Depiction of DNA replication with replication fork, strands and okazaki-fragments. a: template strands, b: leading strand, c: lagging strand, d: replication fork, e: RNA primer, f: Okazaki fragment

• The replication fork is the site on a DNA molecule at which the unwinding of the helices and synthesis of daughter molecules are both occurring.

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• Three and five prime refer to particular carbons on the deoxyribose (5 carbon sugar ring) backbone of DNA
• The 5?-end (pronounced "five prime end") designates the end of the DNA or RNA strand that has the fifth carbon in the sugar-ring of the deoxyribose at its terminus. 
• This is what the phosphate is attached to in the image below.
• The 3?-end of a strand is so named due to it terminating at the hydroxyl group (OH) of the third carbon in the sugar-ring. It is known as the tail end
• DNA replication always occurs from 5' to 3' or from head to tail (or downstream). Nucleic acids can only be synthesized in the 5?-to-3? direction because the polymerase that assembles new strands only attaches new nucleotides to the 3?-hydroxyl (-OH) group
• These terms are relevant to DNA replication.[image]
• [image]note: the deoxyribose is inverted on the three prime to five prime strand.

It must uncoil. This is the task of the enzyme helicase. 

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• Only one daughter cell is given the mutation. The other daughter cell has normal DNA.

• The purpose of the DNA is to give a cell/cells the "recipes" for the protein synthesis that they need to perform in order for the organism to function.

Protein synthesis occurs at the ribosome

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• It must have been read and translated. mRNA advance sequentially in the ribsome. It will not advance before the presenting codon (3 nucleotide bases) has been read and its anticodon (3 base pairs to that sequence) and attending amino acid brought in by tRNA.

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A base pair mutation or a point substition mutation

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A base-pair or a point-substitution mutation.

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• 700- 800 amino acids long. Multiply by 3 to determine the number of base pairs (2100-2400).  The large ones are unfathomably big. 

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1. Nonsense Code
2. Missence Code
3. Silent Mutation

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A nonsense mutation

Think: NO AMINO ACID = NONSENSE

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Missense mutation

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none. 

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a STOP CODE

• In the genetic code, a stop codon (or termination codon) is a nucleotide triplet within messenger RNA that signals a termination of translation.
• Proteins are based on polypeptides, which are unique sequences of amino acids. Most codons in messenger RNA (from DNA) correspond to the addition of an amino acid to a growing polypeptide chain, which may ultimately become a protein. 
• Stop codons signal the termination of this process by binding release factors, which cause the ribosomal subunits to disassociate, releasing the amino acid chain.

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• mRNA can progress no further. A sequence that calls for no amino acid is a stop code. The mRNA stops progressing, and releases the unfinished chain.
• No protein is formed. 

• Nope. It's nonfunctional in this primary structure.
• It needs to fold into its secondary, tertiary or quaternary structure to function
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This is the primary structure of a protein

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• Chains of amino acids, are either coiled into an Alpha Helix or folded into a Beta Pleat  

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• The Alpha Helix and Beta Pleat are secondary protein structures. 
• Many proteins are complete at this level. 
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Hydrogen Bonds

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Proteins, collagen and ligaments are proteins with a secondary structure.

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• The tertiary or quaternary structure of the enzyme, a structure which is created by hydrogen bonds between the component amino acids. 
• e.g. This is superoxide desmutase

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• Hydrogen bonds between specific amino acids in specific relationship with eachother. This is why the "key" end of an enzyme is dependent on a very specific order of amino acid. If the order wrong, the bonds are wrong and the shape is wrong. The "key" won't fit into its "lock".

• It depends on where in the chain the mutation occured.
• If the mutation occurs on the "business end" of the key, no, the enzyme will not function. At least one wouldn't expect it to. 
• Prof. McCleary noted the sticky key that, though bent, still starts the car. 
• An incorrect amino acid will be placed in the polypeptide chain.
• Due to that incorrect amino acid the final structure of the enzyme will be distorted rendering it nonfunctional.
• If the error occurs on the "nonbusiness end" the enzyme may function normally. 

• The location of the mutation or where the error occurs. 
• Generally, if the error occurs in the "key" area, it will be nonfunctional. 
• It may be marginally functional if only a small distortion occurs.
• If it occurs elsewhere, the enzyme may function normally but this is rare.

The code is wrong but the protein is correct.

This is due to the redundancy or degeneracy of DNA

• Redundancy. The genetic code is redundant or degenerate
• Degeneracy of codons is the redundancy of the genetic code, exhibited as the multiplicity of three-codon combinations specifying a single amino acid.

• There are two types of mutations, a point or base pair substituion mutation and a frame shift mutation.

A point mutation or base substitution mutation.

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Nonsense code. No protein created. 

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A missence mutation

(This is the last example in the image below.) 

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a silent mutation (the first point mutation below)

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The  degeneracy or redundency of the genetic code

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• The genetic code is redundent. In this circumstance the incorrect base ended up coding for the same amino acid as the correct base would have, resulting in a normal protein. 

Phenotypic lag. 

• Note: The phenotype is the external manifestation of an organism's genotype. It makes sense that the phenotypic lag is the time it takes for a change in the organism's genome (mutation) to show up in its phenome or the sum of its phenotypic traits. 

• The time between the occurance of a mutation and our ability to visualize or become aware of its presence.

• No. 
• There may be only a single mutant per colony. (1/200) The mutation will not be discernable at this ratio. It would take a much larger number of mutated organisms to make the mutation apparent. 
• The time it takes to reach this number of organisms is the phenotypic lag.

• A mutation arises in the DNA of a single organism. 
• That mutation will be apparent in that organism but the qualities of the individual organism will be masked by the group until the mutated organism has replicated numerous times.

• In order for a mutation to propogated forward, the mutation must equip the organism to be equally successful (or more successful) than its normal counterparts. 

• Not all mutations are propogated forward. 
• Some mutations lead to distinct disadvantages that undercut the competitivness of the organism. Unless the mutation allows the organism to be equally competitive with other organisms in the same media or host, it will not be propogated forward. 

• Nothing. These terms mean exactly the same thing in the context of the genetic code. 

A series of 3 nucleotide bases specify one amino acid. 

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A frame shift muation

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• A gene reads:  ATC TAG CCC AGA
• A mutation results in the following: ATT AGC  CCA GA...
• This is a frameshift mutation as the result of a deletion 
• The C in the first triplet was deleted

• No protein (or an inactive protein) is produced when an organism has a frameshift mutation.
• The entire sequence is skewed from the point of mutation and odds are that a stop code will be encountered before the end of the sequence is reached.  
• The book notes, "In most cases a nonsense code will be encountered, terminating translation."
• Prof. McCleary said that "No one's luck is good enough." 

• The offspring will inherit the faulty DNA and also be unable to produce the given protein. 

• Inborn metabolic errors or inborn errors of metabolism are conditions caused by genetic mutations which result in the absence of or nonfunctionality of some essential element of metabolism, often an enzyme.
• The majority are due to defects of single genes that code for enzymes that facilitate conversion of various substances into others products. 
• In most of the disorders, problems arise due to accumulation of substances which are toxic or interfere with normal function, or to the effects of reduced ability to synthesize essential compounds.

Inborn metabolic errors or inborn metabolic disorders.

NOTE: both diseases result from the inherited inability to produce a given enzyme necessary for the normal metabolic processes of the body. 

• Tay-Sachs disease is a life-threatening disease of the nervous system passed down through families. Tay-Sachs disease occurs when the body lacks hexosaminidase A, a protein that helps break down a chemical found in nerve tissue called gangliosides. Without this protein, gangliosides,  build up in cells, especially nerve cells in the brain, killing the cells and resulting in death. 
• Phenylketonuria (PKU) is an autosomal recessive metabolic genetic disorder characterized by a mutation in the gene for the hepatic enzyme phenylalanine hydroxylase (PAH), rendering it nonfunctional. This enzyme is necessary to metabolize the amino acid phenylalanine (Phe) to the amino acid tyrosine. Untreated PKU can lead to intellectual disability, seizures, and other serious medical problems.

• The social taboos which has perpetuated diseases like Tay-Sachs are the taboos which say one must not marry or have children with anyone outside their own culture & ethnic background.
• This limits the genetic pool resulting in an increased likelihood that two parents will carry the same mutation. 
• The more exclusive the population, the higher the incidence of mutation
• [image]

• Tay-Sachs would be much, much less common in bacteria because bacteria, being haploid, would not be able to mask the presence of the mutated gene with a normal allele. 
• The disease, therefore, would manifest in the parent cell which would die before "reproductive age" as the children who have the actual disease do. 
• The disease would not be perpetuated

• Because bacteria are haploid, their mutations are not masked. Therefore, in order for a mutation to be propogated forward it must help the organism in some way, or give it an at-least-equally competative edge with its nonmutated counterpart.

• Spontaneous mutations are relatively infrequent considering the number of cells and cell turnover in a multicellular organism.

• Many antibiotic resistant strains have resulted from:
• a. raditation exposure
• b. chemical exposure
• c. spontaneous mutation
• d. all of the above

• Nope.
• It's not likely that two organisms will mutate in the same way and end up with the same antibiotic resistance. 
• e.g. Entero coccus and Staph aureous are both vancomycin resistant. 
• It is highly unlikely that both organisms mutated in the same spot.
• (Conjugation is the more likely means of promotion)

• Radiation often fragments chromosomes. The chromosomes then reanneal in a "funky fashion"

• Chemically induced mutations are more predictable in their results than radiation induced mutations.

• The two types of induced mutations are radiation induced and chemically induced.
• Radiation induced mutations are less predictable in their result. 

• Chemical mutations allow more specific predications because each chemical interacts with the DNA in a highly specific way. 

 By performing the Ames Test

• Note: The Ames test reviewed in lecture differs from that in our textbook. The "Ames test" as discussed in lecture is very similiar to the replica plating on p. 229, in that it uses a wildtype salmonella capable of producing its own histadine. 
• The actual Ames test uses a histadine dependent strain and determines mutation by the  reversion in the salmonella's ability to produce histadine.
• That is, a mutagenic agent will apparently make this strain of salmonella able to produce its own histadine. 

Salmonella, specifically Salmonella typhimurium

• Despite the fact that we reviewed an Ames test that used wild type salmonella, Prof. did note that this was the actual type used. 
• She also noted that we did not need to know the strain, just that it was a type of Salmonella. 

• An essential amino acid is an amino acid which the organism must take in from a nutrient source
• An amino acid which is not produced by the organism and must be supplied by the diet. 

• A nonessential amino acid is an amino acid which, given a spectrum of amino acids, the body can  make for itself.
• A nonessential amino acid does not need to be obtained through the diet. 

• For wild-type salmonella histidine is a nonessential amino acid. That is, wild-type salmonella does not need to get this amino acid from its environment/diet, it makes it itself. 

Histidine is employed in the Ames test.

Histidine is a nonessential amino acid to wild type salmonella

• In the Ames test (lecture version) wild-type salmonella and chemical (X) whose mutagenicity is to be determine, are added to a broth culture. 
• In the book, an auxotrophic version of salmonella is used in the Ames test. 

• It must be incubated so that the organism grows & metabolizes in the presence of the chemical. 

• In the lecture version of the Ames test a chemical (x) is mixed with wild-type salmonella in a broth culture. This mixture is incubated. 
• This mixture is then poured onto two plates: one containing the amino acid histidine and one containing no histidine. 

• Both plates contain a growth medium. One plate contains the amino acid histidine. One plate contains no histidine. 

• Growth on ONLY the histidine containing plate indicates that chemical X is a mutagen. 
• This is for a version of the test which uses wild type salmonella, for which histidine is a nonessential amino acid. 
• Growth on only the histidine containing plate would indicate that the organism could no longer produce its own histidine and a mutation had occurred. 
• If this same test were performed with a histadine depedendent strain of salmonella, [an auxotrophic strain], growth on both plates would indicate a positve result, or that chemical X is mutagenic. 
• Reversion to the ability to produce histadine indicates mutation. 
• This is what the textbook version of the Ames test shows. 

• On the Ames test, (as reviewed in lecture- using wild-type salmonella) a negative result is indicated by growth on both the plate containing histidine and the plate without histidine. 
• This would suggest that the organism exposed to chemical x is still capable of producing its own histidine and no mutation has occured. 
• On the Ames test, according to the text book, [using an auxotrophic form of salmonella] a negative result would be indicated by growth on only the histadine containing plate. 

• According to the lecture version of the Ames test, growth on both plates indicated a negative result, or that: "Chemical X is not mutagenic"

• According to the lecture on the Ames test*, growth on the histidine plate only indicates a positive result: "Chemical X is mutagenic"
• Wild-type salmonella, the bacteria used in the lecture version,  normally produces its own histidine. This result indicates that the bacteria lost its ability to do this, indicating mutation. 
• * Note: The lecture version differs significantly from the text book version. 

The Ames test.

• Note: This is actually used in replica plating, which is distinct from the Ames test. It seems like the Ames test in our lecture was a cross between replica plating and the classic Ames test. 

• Replica plating is a technique for testing the genetic characteristics of bacterial colonies. A dilute suspension of bacteria is spread, in a petri dish, on agar containing a medium expected to support the growth of all bacteria, the master plate. 
• Each bacterial cell in the suspension is expected to give rise to a colony. A sterile velvet pad, the same size as the petri dish, is then pressed onto it, picking up a sample of each colony. 
• The bacteria can then be stamped onto new sterile petri dishes in the identical arrangement. 
• The media in the new plates can be made up to lack specific nutritional requirements or to contain antibiotics. Thus colonies can be identified that cannot grow without specific nutrients or that are antibiotic resistant and cells with mutations in particular genes can be isolated. 
• from: biology-online.org

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• Growth on both plates indicates a negative result, chemical X is not mutagenic.
• The wild type salmonella was able to grow without utilizing the salmonella supplied by the plate, indicating it had not mutated

• According to lecture, growth on only the histidine positive plate indicates a positive result on the Ames test, chemical X is mutagenic. This is conditional on the organism being wild type salmonella, not auxotrophic salmonella. 

auxotrophs or an auxotrophic form of salmonella or histidine auxotrophs.

or [image]

• An auxotroph is an organism that has a nutritional requirement not found in the parent strain.

• A nutrient required by the auxotrophic form of an organism, that is, an organism that has mutated from its parent stain and as a result has a nutritional requirement not seen in the parent strain. 

• In the Ames test:
• the mutated salmonella is the auxotrophic form
• histidine becomes conditionally essential

• Normally produced by the organism, in certain circumstances  a conditionally essential nutrient must be supplied by an outside source in order for the organism to survive and thrive? 

• All chemicals that are used in human compounds, whether for ingestion or for cosmetic purposes must pass an Ames test prior to FDA approval 

• Mutation is almost excluisvely a vertical process.
• [image]
• During DNA replication a mutation occurs.
• During binary fission, the mutation is passed on to one of the organism's daughter cell. 
• That cell divides resulting in two mutated offspring.

• In order for conjugation to occur the donor organism must have a pilus and plasmid. 

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• A pilus is used by a donor bacteria to dock with a recipient bacteria in order to facilitate the transfer of plasmid based information during conjugation.

Transformation

• Prof. McCleary used the metaphor of a bag lady finding a coat and putting it on to describe the process by which bacteria take in :naked" DNA, or DNA from a dead bacterium, and incorporate it into their own DNA.

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• When bacterial cells die, they lose their integrity. The inside can no longer be contained by the cell wall and "everything becomes a big soup pot". 
• This includes the now naked DNA which can be taken up by bacteria capable of transformation. 
• [image]

• The bacteria capable takes up the components of the dead bacteria: 
• the neuleotide bases, the amino acid pool, the deoxyribose and ribose. 
• it also takes up complete genes if present. 

• Transformation
• Think of the "bag lady" coming across an elegant coat amid a pile of discarded rubbish. She puts on the coat and is "transformed". 
• This is akin to the bacteria coming across a gene for antibiotic resistance in the debris of dead bacteria. 

YES! Transformation contributes to the rise of antibiotic resistance.

• Bacteria capable of transformation in the presence of dead bacteria may enounter in those remains a gene for antibiotic resistance. 
• They may encorporate that gene into their own genome and pass it on to their offspring.  

• That is where virulent bacteria will enounter other virulent bacteria.
• Any means of genetic transfer or recombination: Conjugation, transformation, transduction, or specialized transduction can result in the virulence factors from one organism being transferred to another organism, including antibiotic resistance. 

The transfer of genetic information using a virus.

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Transduction 

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Bacteriophages or phages

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• Bacteriophages or phages are viruses that infect bacteria 
• Bacteriophages are responsible for transduction, the transfer of genetic information from one bacterium to another using a virus. 
• Bacteriophages are also responsible for specialized transduction,  in which only a few specific genes from one bacterial cell are transferred to the second bacterial host by a phage

A bacteriophage and a [competent] bacteria

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• When viruses infect any living cell they will release their nucleic acid and then utilize the parts of the host cell to copy their own genetic code.

• We were given a rather rough outline of the process. It was not entirely in line with the information in the text book. 
• This information is, for the most part, from the book. 
• Information specific to class is in brackets. 
1. A bacteriophage injects its genome into a [competent] bacteria, [getting rid of its outer coat]
2. Phage DNA and proteins are made.
3. An enzyme in the phage breaks down the bacteria's DNA. [Prof. M said that the phage DNA is inserted directly into the bacterial DNA. This is not in the textbook.]
4. New phage DNA is packaged in viral protein coats, also made using the bacteria's materials. 
5. Some of the broken down bacterial DNA may end up in a phage protein coat. This is an accidental and RANDOM occurance. 
6. When new bacteriophages are released from the host cell,  bacteriophages inject that bacterial DNA [combined with viral according to lecture] into another bacteria.
7. That transduced bacterial DNA becomes integrated into the host bacterium's chromosome by recombination.
• This is the genetic transfer process of generalized transduction. 

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Specialized transduction

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• The textbook mentions only transformation. The lecture mentioned only transduction. I aksed Prof. M. She said both require that the bacteria involved has special properties, or competence. 

• According to lecture, generlized transduction is more likely to result in a viral infection becase in specialized transduction not all of the necessary viral DNA has been copied. 
• The book indicates something different. It's a little puzzling.