examprepare

Chapter 6 review microbiology

1, Know the differences between prokaryotic and eukaryotic cells and chromosomes
Cell's ------
The major similarities between the two types of cells (prokaryote and eukaryote) are:

1. They both have DNA as their genetic material.
2. They are both membrane bound.
3. They both have ribosomes .
4. They have similar basic metabolism .
5. They are both amazingly diverse in forms.

The major differences :
1. eukaryotes have a nucleus and membrane-bound organelles , while prokaryotes do not.The DNA of prokaryotes floats freely around the cell. the DNA of eukaryotes is held within its nucleus.The organelles of eukaryotes allow them to exhibit much higher levels of intracellular division of labor than is possible in prokaryotic cells
2. Eukaryotic cells are, on average, ten times the size of prokaryotic cells.
3. Genomic composition and length: The DNA of eukaryotes is much more complex and therefore much more extnsive than the DNA of prokaryotesEukaryotic DNA is linear; prokaryotic DNA is circular (it has no ends)
4. Prokaryotes have a cell wall composed of peptidoglycan, a single large polymer of amino acids and sugar . Many types of eukaryotic cells also have cell walls, but none made of peptidoglycan
5. Eukaryotic DNA is complexed with proteins called "histones," and is organized into chromosomes; prokaryotic DNA is "naked," meaning that it has no histones associated with it, and it is not formed into chromosomes. Though many are sloppy about it, the term "chromosome" does not technically apply to anything in a prokaryotic cell. A eukaryotic cell contains a number of chromosomes; a prokaryotic cell contains only one circular DNA molecule and a varied assortment of much smaller circlets of DNA called "plasmids." The smaller, simpler prokaryotic cell requires far fewer genes to operate than the eukaryotic cell

2, What is a genotype? What is a phenotype?

genotype is the specific genetic makeup (the specific genome) of an individual, usually in the form of DNA. Together with the environmental variation that influences the individual, It codes for the phenotype of that individual. the sepcific set of genes an organism posseses. Two alleles type for each gene. Hyterozygous: difference alleles: Bb. Homozygous: similiar alleles: BB, bb.

phenotype of an individual organism is either its total physical appearance and constitution or a specific manifestation of a trait, such as size or eye color, that varies between individuals. it is the collection of characteristics of an organism that an investigator can observe. Gene expression and result of transcription and translation.

3. Know the structure and function of both DNA and RNA.
DNA:
1.DNA is composed of purine and pyrimidine nucleosides that contains the sugar deosyribose and a phosphate group.
2. A double helix structure consisting two chains of nucleosides coiled around each other.
3. The purine: adnine and guanine. the primidine: thymine and cytosine.
4. two strand are not positioned directing opposite one another. therefore a major groove and smaller minor groove are formed during helix backbone.
5. the two polynucleotide chains are antiparallel.

RNA:

1. The sugar in the RNA molecule is ribose. DNA's sugar is deoxyribose.
2. RNA is usually a single stranded molecule while DNA is nearly always double stranded.
3. DNA's rigid double helix structure allows for only one function (information storage) whereas RNA's greater molecular diversity results in a wider range of functions
4. RNA uses the nucleotide uracil instead of thymine
5. DNA is often 10³ to 10⁶ times larger than RNA
6. RNA is much less stable than DNA. As a single stranded molecule it has no way of reparing itself.

4. What were the historical experiments that led to the paper on the structure of DNA? Who wrote it?

1. Frederick Griffith(1928) demonstrated the phenomenon of transformation: nonvirulent bacteria could become virculent when live, non virulent bacteria were miced with dead, virulent bacteria.
2. Avery, Macleod, and McCarty (1944) demonstrated that the transforming principle was DNA.
3. Hershey and Chase (1952) showed that for the T2 bacteriophage, only the DNA was needed for infectivity. therefore they proved that DNA was the genetic material.
4. Erwin Chargaff: show that in natural DNA the number of guanine units equals the number of cytosine units and the number of adenine units equals the number of thymine units
5. Rosalind Franklin: obtained some excellent x-ray diffraction photographs of DNA' using the recent technique of the time called x-ray crystallography.

The double helix is the structure of DNA as first published by James D. Watson and Francis Crick in 1953. They constructed a molecular model of DNA in which there were two complementary, antiparallel (side-by-side in opposite directions) strands of the bases guanine, adenine, thymine, and cytosine, covalently linked through phosphodiester bonds. The four nitrogen-containing bases found in DNA are divided into two groups: purines and pyrimidines. Two-ringed bases are purines. One-ringed bases are called pyrimidines. Adenine and Guanine are purines, whilst Thymine and Cytosine are Pyrimidines.

5. How is DNA replicated? What enzymes are needed? Where does DNA polymerase function? What does DNA ligase do? What is Theta replication? What are replication forks?

1. DNA is replicated by uncoiling of the helix, strand separation by breaking of the hydrogen bonds between the complementary strands, and synthesis of two new strands by complementary base pairing.Replication begins at a specific site in the DNA called the origin of replication.

2. DNA replication is bidirectional from the origin of replication. unwinding enzymes called DNA helicasescause the two parent DNA strands to unwind and separate from one another at the origin of replication to form two "Y"-shaped replication forks(The actual site of DNA replication where free deoxyribonucleotides hydrogen bond with the nucleotides on each unwound parent DNA strand.)

3. These replication forks are the actual site of DNA copying.Helix destabilizing proteins bind to the single-stranded regions so the two strands do not rejoinEnzymes called topoisimerases produce breaks in the DNA and then rejoin them in order to relieve the stress in the helical molecule during replication.

4. As the strands continue to unwind and separate in both directions around the entire DNA molecule, new complementary strands are produced by the hydrogen bonding of free DNA nucleotides with those on each parent strand.As the new nucleotides line up opposite each parent strand by hydrogen bonding, enzymes called DNA polymerases join the nucleotides by way of phosphodiester bonds. In the end, each parent strand serves as a template to synthesize a complementary copy of itself, resulting in the formation of two identical DNA molecules.

5. one parent strand - the one running 3' to 5' and called the leading strand (def) - can be copied directly down its entire length (see Fig. 17). However, the other parent strand - the one running 5' to 3' and called the lagging strand (def) - must be copied discontinuously in short fragments (Okazaki fragments) of around 100-1000 nucleotides each as the DNA unwinds.During this process, Finally, the DNA fragments themselves are hooked together by the enzyme DNA ligase.

6. the DNA molecules may attach to the cytoplasmic membrane and, as the cell elongates, the two DNA molecules are physically separated

Theta replication: DNA replication of prokaryotes.

6. Compare the structure of plasmids in bacterial cells to the structure of the bacterial chromosome. What is the role of plasmids in bacterial cells?

Prokaryotic cells (bacteria) contain their chromosome as circular DNA. Usually the entire genome is a single circle, but often there are extra circles called plasmids. The DNA is packaged by DNA-binding proteins. Plasmids are typically circular dsDNA molecules which range in size from 2 Kb to 100 Kb. These plasmids will be supercoiled in the cell

plasmid is an extra-chromosomal element, often a circular DNA. Plasmids are small molecules of double stranded, helical, nonchromosomal DNA.lasmids usually contain between 5 and 100 genes. Plasmids are not essential for normal bacterial growth and bacteria may lose or gain them without harm. They can, however, provide an advantage under certain environmental conditions. For example, under normal environmental growth conditions, bacteria are not usually exposed to antibiotics and having a plasmid coding for an enzyme capable of denaturing a particular antibiotic is of no value. three important elements:

• An origin of replication:Since a plasmid is (by definition) an extrachromosomal element, it cannot make use of any origin of DNA replication in a chromosome. That is, DNA synthesis within (i.e. copying of) a plasmid depends on its having an origin of DNA synthesis of its own. Obviously, if a plasmid couldn't be copied, it would be rapidly diluted out in a population of dividing cells because it couldn't be passed on to daughter cells
• A selectable marker gene (e.g. resistance to ampicillin):carrying a plasmid puts a cell at a selective disadvantage compared to its plasmid-free neighbors, so the cells with plasmids grow more slowly. Cells that happen to "kick out" their plasmid during division may be "rewarded" by having a higher rate of growth, and so these plasmid-free (sometimes referred to as "cured") cells may take over a population. If a plasmid contains a gene that the cell needs to survive (for example, a gene encoding an enzyme that destroys an antibiotic), then cells that happen to kick out a plasmid are "punished" (by subsequent death) rather than "rewarded" (as in the previous scenario). That selective pressure helps to maintain a plasmid in a population.
• A cloning site (a place to insert foreign DNAs)
• 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 code for synthesis of a few proteins not coded for by the nucleoid.

7. support the statement "DNA replication if semiconservtive"
8.State the central dogma of protein synthesis.
DNA-------(transcription)-------RNA--------(translation)--------Protein.
Transcription is the making of an RNA molecule off a DNA template. Translation is the construction of an amino acid sequence (polypeptide) from an RNA molecule.

9. three major types of RNA( produced by transcription)
mRNA: messenger RND carries the message to encode protein in a condon.
rRNA: a component of the ribosomes
tRNA: anti-codon: transfer RNA

10. What controls the expression of genes? What is an operon? Remember that the enzyme in bacteria that degrades lactose is ß-galactosidase, not lactase (lactase works in larger organisms)
http://www.emc.maricopa.edu/faculty/farabee/biobk/BioBookGENCTRL.html
http://www.sumanasinc.com/webcontent/anisamples/majorsbiology/lacoperon.html

11. What were the experiments of Jacob and Monod? How does the lac operon work? What are the promotor, or operator sites on DNA, which molecule is the repressor, or inducer? Explain
promoter is a DNA sequence that enables a gene to be transcribed. The promoter is recognized by RNA polymerase, which then initiates transcription.

• RNA polymerase I: transcribes genes encoding ribosomal RNA
• RNA polymerase II: transcribes genes encoding messenger RNA and certain small nuclear RNAs
• RNA polymerase III: transcribes genes encoding tRNAs and other small RNAs

An operator is a segment of DNA that regulates the activity of the structural genes of an operon it is linked to, by interacting with a specific repressor or activator. It is a regulatory sequence for shutting a gene down or turning it "on".In negative inducible operons, a regulatory repressor protein is bound to the operator and it prevents the transcription of the genes on the operon. If an inducer molecule is present, it binds to repressor and changes its conformation so that it is unable to bind to the operator. This allows for the transcription of the genes on the operator. In negative repressible operons, transcription of the genes on the operon normally takes place. Repressor proteins are produced by a regulator gene but they are unable to bind to the operator in their normal conformation. However repressor molecules can bind to the repressor protein and change its conformation so that it can bind to the operator. The activated repressor proteins bind to the operator and prevent transcription.
A repressor is a DNA-binding protein that regulates the expression of one or more genes by decreasing the rate of transcription. This blocking of expression is called repression.Repressor proteins are coded for by regulator genes. Repressor proteins then attach to a DNA segment known as the operator. By binding to the operator, the repressor protein prevents the RNA polymerase from creating messenger RNA.

12. What is mutation? How does it occur?name three mutagens and its effects on bacterial DNA.
Mutation: An error during DNA replication that results in a change in the sequence of deoxyribonucleotide bases in the DNA.
Spontaneous mutation (def) occurs naturally (a normal mistake rate) about one in every million to one in every billion divisions and is probably due to low level natural mutagens normally present in the environment. Induced mutation (def) is caused by mutagens, substances that cause a much higher rate of mutation.
Spontaneous mutation :

1. Mechanisms of mutation

a. Substitution of a nucleotide (point mutations (def)): substitution of one deoxyribonucleotide for another during DNA replication (see Fig. 1). This is the most common mechanism of mutation. Substitution of one nucleotide for another is a result of tautomeric shift, a rare process by which the hydrogen atoms of a deoxyribonucleotide base move in a way that changes the properties of its hydrogen bonding. For example, a shift in the hydrogen atom of adenine enables it to form hydrogen bonds with cytosine rather than thymine. Likewise, a shift in the hydrogen atom in thymine allows it to bind with guanine rather than adenine.

b. Deletion or addition of a nucleotide (frameshift mutations (def)): deletion or addition of a deoxyribonucleotide during DNA replication (see Fig. 2 and Fig. 3).

2. Results of mutation

One of four things can happen as a result of these mechanisms of mutation and the resulting change in the deoxyribonucleotide base sequence mentioned above:

a. A missense mutation (def) occurs. This is usually seen with a single substitution mutation and results in one wrong codon (def) and one wrong amino acid (see Fig. 4).

b. A nonsense mutation occurs (def). If the change in the deoxyribonucleotide base sequence results in transcription (def) of a stop or nonsense codon (def), the protein would be terminated at that point in the message (see Fig. 5).

c. A sense mutation (def) occurs. This is sometimes seen with a single substitution mutation when the change in the DNA base sequence results in a new codon still coding for the same amino acid (see Fig. 6). (With the exception of methionine, all amino acids are coded for by more than one codon.)

d. A frameshift mutation occurs (def). This is seen when a number of DNA nucleotides not divisible by three is added or deleted. Remember, the genetic code is a triplet code where three consecutive nucleotides code for a specific amino acid. This causes a reading frame shift and all of the codons and all of the amino acids after that mutation are usually wrong (see Fig. 7); frequently one of the wrong codons turns out to be a stop or nonsense codon and the protein is terminated at that point.

b. Induced mutation (def) is caused by mutagens, substances that cause a much higher rate of mutation.

Chemical mutagens generally work in one of three ways.

• Some chemical mutagens, such as nitrous acid and nitrosoguanidine work by causing chemical modifications of purine and pyrimidine bases that alter their hydrogen-bonding properties. For example, nitrous acid converts cytosine to uracil which then forms hydrogen bonds with adenine rather than guanine.
• Other chemical mutagens function as base analogs. They are compounds that chemically resemble a nucleotide base closely enough that during DNA replication, they can be incorporated into the DNA in place of the natural base. Examples include 2-amino purine, a compound that resembles adenine, and 5-bromouracil, a compound that resembles thymine. The base analogs, however, do not have the hydrogen-bonding properties of the natural base.
• Still other chemical mutagens function as intercalating agents. Intercalating agents are planar three-ringed molecules that are about the same size as a nucleotide base pair. During DNA replication, these compounds can insert ir intercalate between adjacent base pairs thus pushing the nucleotides far enough apart that an extra nucleotide is often added to the growing chain during DNA replication. An example is ethidium bromide.

Chemical mutagens can also activate what is called SOS repair that can lead to further mistakes in DNA base pairing (see below).

Certain types of radiation can also function as mutagens.

• Ultraviolet Radiation. The ultraviolet portion of the light spectrum includes all radiations with wavelengths from 100 nm to 400 nm. It has low wave length and low energy. The microbicidal activity of ultraviolet (UV) light depends on the length of exposure: the longer the exposure the greater the cidal activity. It also depends on the wavelength of UV used. The most cidal wavelengths of UV light lie in the 260 nm - 270 nm range where it is absorbed by nucleic acid.

  In terms of its mode of action, UV light is absorbed by microbial DNA and causes adjacent thymine bases on the same DNA strand to covalently bond together, forming what are called thymine-thymine dimers (see Fig. 8). As the DNA replicates, nucleotides do not complementary base pair with the thymine dimers and this terminates the replication of that DNA strand. However, most of the damage from UV radiation actually comes from the cell trying to repair the damage to the DNA by a process called SOS repair. In very heavily damaged DNA containing large numbers of thymine dimers, a process called SOS repair is activated as kind of a last ditch effort to repair the DNA. In this process, a gene product of the SOS system binds to DNA polymerase allowing it to synthesize new DNA across the damaged DNA. However, this altered DNA polymerase loses its proofreading ability resulting in the synthesis of DNA that itself now contains many misincorporated bases. (Most of the chemical mutagens mentioned above also activate SOS repair.)

• Ionizing Radiation. Ionizing radiation, such as X-rays and gamma rays, has much more energy and penetrating power than ultraviolet radiation. It ionizes water and other molecules to form radicals (molecular fragments with unpaired electrons) that can break DNA strands and alter purine and pyrimidine bases.

13. Describe the ways that bacteria can repair DNA damage or replication errors
damage reversal--simplest; enzymatic action restores normal structure without breaking backbone 1. This is one of the simplest and perhaps oldest repair systems: it consists of a single enzyme which can split pyrimidine dimers (break the covalent bond) in presence of light. 2. X-rays and some chemicals like peroxides can cause breaks in backbone of DNA. Simple breaks in one strand are rapidly repaired by DNA ligaseMicrobial mutants lacking ligase tend to have high levels of recombination since DNA ends are recombinogenic (very reactive).

damage removal--involves cutting out and replacing a damaged or inappropriate base or section of nucleotides. 1. base excision repair: The damaged or inappropriate base is removed from its sugar linkage and replaced. These are glycosylase enzymes which cut the base-sugar bond. example: uracil glycosylase--enzyme which removes uracil from DNA. The enzyme recognizes uracil and cuts the glyscosyl linkage to deoxyribose.The sugar is then cleaved and a new base put in by DNA polymerase using the other strand as a template. Mutants lacking uracil glycosylase have elevated spontaneous mutation levels (C to U is not fixed, which leads to transitions) and are hyper-sensitive to killing and mutation by nitrous acid (which causes C to U deamination). 2. mismatch repair: This process occurs after DNA replication as a last "spellcheck" on its accuracy. In E. coli, it adds another 100-1000-fold accuracy to replication. It is carried out by a group of proteins which can scan DNA and look for incorrectly paired bases (or unpaired bases) which will have aberrant dimensions in the double helix. The incorrect nucleotide is removed as part of a short stretch and then the DNA polymerase gets a second try to get the right sequence. 3. nucleoride excision repair: This system works on DNA damage which is "bulky" and creates a block to DNA replication and transcription (so--UV-induced dimers and some kinds of chemical adducts). It probably recognizes not a specific structure but a distortion in the double helix. The mechanism consists of cleavage of the DNA strand containing the damage by endonucleases on either side of damage followed by exonuclease removal of a short segment containing the damaged region. DNA polymerase can fill in the gap that results.

damage tolerance--not truly repair but a way of coping with damage so that life can go on. 1.recombinal repair This is a repair mechanism which promotes recombination to fix the daughter-strand gap--not the dimer--and is a way to cope with the problems of a non-coding lesion persisting in DNA.his type of recombinational repair is generally accurate (although it can cause homozygosis of deleterious recessive alleles) and requires a homolog or sister chromatid. 2. A second type of recombinational repair which is used primarily to repair broken DNA ends such as are caused by ionizing radiation and chemical mutagens with similar action is the non-homologous end-joining reaction. 3. mutagenic repair.An alternative scenario for a DNA polymerase blocked at a dimer is to change its specificity so that it can insert any nucleotide opposite the dimer and continue replication ("mutate or die" scenario).

14. What is a transposon? Who did the research and why is it important?

Transposons are sequences of DNA that can move around to different positions within the genome of a single cell, a process called transposition. In the process, they can cause mutations and change the amount of DNA in the genome. Transposons are also called "jumping genes" or "mobile genetic elements". Discovered by Barbara McClintock early in her career, the topic went on to be a Nobel winning work in 1983.

1. class I: Retrotransposons work by copying themselves and pasting copies back into the genome in multiple places. Initially retrotransposons copy themselves to RNA (transcription) but, in addition to being translated, the RNA is copied into DNA by a reverse transcriptase (often coded by the transposon itself) and inserted back into the genome.

2. Class II transposons move by cut and paste, rather than copy and paste, using the transposase enzyme. Different types of transposase work in different ways. Some can bind to any part of the DNA molecule, and the target site can therefore be anywhere, while others bind to specific sequences. The transposase then cuts the target site to produce sticky ends, cuts out the transposon and ligates it into the target site, and then fills in the sticky ends with the corresponding base pairs.

15.What is replica plating –how does it work – and why is it important in identifying bacterial mutants?

replica plating is a technique in which one or more secondary Petri plates containing different solid (agar-based) selective growth media (lacking nutrients or containing chemical growth inhibitors such as antibiotics) are inoculated with the same colonies of microorganisms from a primary plate (or master dish), reproducing the original spatial pattern of colonies. The technique involves pressing a velvet-covered disk to a primary plate, and then imprinting secondary plates with cells in colonies removed by the velvet from the original plate. Generally, large numbers of colonies (roughly 30-300) are replica plated due to the difficulty in streaking each out individually onto a separate plate.

The purpose of replica plating is to be able to compare the master plate and any secondary plates to screen for a selectable phenotype. For example, a colony which appeared on the master plate but failed to appear at the same location on a secondary plate shows that the colony was sensitive to a substance on that particular secondary plate. Common screenable phenotypes include auxotrophy and antibiotic resistance.

Replica plating is especially useful for negative selection. For example, if one wanted to select colonies that were sensitive to ampicillin, the primary plate could be replica plated on a secondary Amp⁺ agar plate . The sensitive colonies on the secondary plate would die but the colonies could still be deduced from the primary plate since the two have the same spatial patterns from ampicillin resistant colonies. The sensitive colonies could then be picked off from the primary plate.By increasing the variety of secondary plates with different selective growth media, it is possible to rapidly screen a large number of individual isolated colonies for as many phenotypes as there are secondary plates.

16. What is the Ames test and why is it useful and important? What microbe is involved?

to test for mutagenic properties of a chemical compound. A compound is said to be mutagenic if it causes a change in the DNA (deoxyriboneucleic acid) of a living cell or organism. The test is named after its inventor, Bruce Ames.

using strains of bacteria, generally Escherichia coli or Salmonella typhimurium that already have a single mutation, for example, a strain that cannot produce histidine, an amino acid that is essential for the bacterium to grow if not provided externally with essential nutrients. Cultures of the bacteria are grown in an agar containing dish so that a "lawn" of bacteria is present.

The experimental cultures are exposed to the agent to be tested while the positive control cultures are exposed to a known mutagen to confirm that there has been no contamination of the strain. Strains of bacteria are available which have been genetically modified such that only a certain type of mutation (i.e. a base pair mutation or a frameshift mutation) will cause the strand to revert to a normal state, not requiring nutrients to grow. If the mutation screened for has in fact occurred, dense spots in the colonies will form. A certain number of spots may form due to random mutation not caused by the agent; therefore, data analysis using control dishes is necessary. Occasionally a tested agent will be toxic enough to simply kill the bacterial culture in which case a "thin lawn" is observed.

posted by C-T @ 5:12 PM