Microbio Ch.8 - Part 2

Restriction Endonuclease- other examples

*review this ligase, reverse transcriptase, complementary DNA (cDNA)

Transposons can result in (3)

1) changes in traits such as colony morphology, pigmentation, and antigenic characteristics 2) replacement of damaged DNA 3) intermicrobial transfer of drug resistance (in bacteria)

Mutation vs Recombination

Mutation: occurs when changes occur to nucleotide sequence Recombination: occurs when whole segments of genetic info are transferred between organisms

Restriction Fragments

pieces of DNA produced by restriction endonucleases

Generalized Transduction

- Bacteriophage serves as a carrier from a donor cell to a recipient cell - Random fragments of disintegrating host DNA are taken up by the bacteriophage - Any gene from the bacterium can be transmitted

Gram Negative Conjugation

- F factor, refers to the donor's plasmid, allows for the synthesis of a conjugative pilus - recipient cell has a recognition site on its surface - F+ cell that has the plasmid, F- cell that lacks the plasmid - contact is made when a pilus grows out from the F+ cell, attaches to the surface of the F- cell, contracts, and draws the two cells together - F factor (plasmid) is copied, and copy is transferred to F- cell

Specialized Transduction

- Highly specific part of the host genome is regularly incorporated into the virus - *Explained by the prior existence of a temperate prophage inserted in a fixed site on the bacterial chromosome* - When activated, prophage DNA separates from the bacterial chromosome, carrying a small segment of host genes with it - During the lytic cycle, these specific viral-host gene combinations are incorporated into the viral particles and carried to another bacterial cell

Transposons

- a small piece of DNA integrated into host cell chromosome, aka "jumping gene", capable of shifting from one part of the genome to another (i.e. from chromosome to plasmid, plasmid to chromosome, or to another spot along a chromosome) - allows for a 4th type of genetic transfer - transposon can excise itself and move from one location to another in genome, or it can replicate prior to moving

Excision Repair of Mutations

- enzymes break the bonds between the bases and the sugar-phosphate strand at the site of the error - a different enzyme removes the defective bases one at a time - the remaining gap is filled in by DNA polymerase I and ligase

Transformation Details

- facilitated by DNA-binding proteins on the cell wall, though requires no special appendages (thus, indirect) - recipient must be competent - capable of accepting genetic material - nonspecific - can occur between 2 foreign organisms (not of same species) - Useful for recombinant DNA technology, in which genes from a completely unrelated organism are inserted into a plasmid, plasmid is introduced into a competent bacterial cell (can be carried out in a test tube!)

Repair of UV Damage

- photoactivation/light repair through photolyase - successful only for a small number of UV mutations - cells cannot repair severe, widespread damage and will die

Cloning Vector: Plasmid

- small, well characterized, easy to manipulate - can be transferred into appropriate cells through transformation*

E.Coli F-Factor Transfer Details

- the donor (F+) cell makes a copy of its F factor (plasmid) - transmits this to a recipient (F-) cell - turns it into an F+ cell capable of producing a pilus and conjugating with other cells

Depending on mode of Transmission, there are 3 means of genetic recombination:

1) Conjugation (direct) 2) Transformation (indirect) 3) Transduction (indirect)

E. Coli Conjugation - 2 Methods

1) F-Factor Transfer 2) High Frequency recombination

What is mean by the term "Conservative" process, in reference to Conjugation?

Conjugation is a conservative process in which the donor bacterium retains ("conserves") a copy of the genetic material being transferred

What is the proofreading mechanism in DNA, used to repair mistakes in replication?

DNA Polymerase III and I

Technical Aspects of Recombinant DNA and gene cloning

DNA is removed from cells and separated into fragments by endonucleases - fragments are inserted into vectors and cloned - cloned fragments are probed for desired sequences - Gene is synthesized from isolated mRNA transcripts using reverse transcriptase - Gene is amplified using PCR

PCR: Primers

DNA strands 15 - 30 bases long that serve as landmarks where DNA amplification should begin!

Most spontaneous mutations are beneficial

F. Most are NOT beneficial. Only a small number of variations create variant strains that more readily adapt, survive, and reproduce

Transduction: 2 forms

Generalized and Specialized

Polymerase Chain Reaction (PCR)

Rapidly increases the amount of DNA in a sample without the need for making cultures or carrying out complex purification techniques! • Sensitive enough to detect cancer from a single cell or diagnose an infection from a single gene copy! • Rapid enough to replicate target DNA from a few copies to billions of copies in a few hours!

Mutant Strain

Shows Variance in one or more of the following: - morphology - nutritional characteristics! - genetic control mechanisms! - resistance to chemicals! - temperature preference! - enzymatic function

Causes of Mutations: Spontaneous vs Induced

Spontaneous mutation: a random change in the DNA arising from errors in replication Induced mutations: result from exposure to known *mutagens*, which are primarily physical or chemical agents that disrupt DNA - radiation: UV light, X rays - chemicals: nitrous acid

Acquired drug resistance is a clear model for this type of environmental change, resulting in selection and adaptation

T

As long as the environment is stable, mutants only comprise a small percentage of the population. When the environment changes, some mutants will be equipped to survive in the new environment

T

Mutations are permanent and heritable and will be passed on to the offspring of organisms and viruses

T

T/F Primary intent of Recombinant DNA Tech is to deliberately remove genetic material from one organism and combine it with that of a different organism

T. Bacteria can be genetically engineered to mass produce substances such as hormones, enzymes, and vaccines difficult to synthesize by usual industrial methods!

Wild Type

a microorganism that exhibits a natural, nonmutated characteristic

How Mutation Effects the Cell

a permanent change in the DNA that is transcribed into mRNA and translated can change structure and function of a protein, and thus, change the physiology of a cell. some changes are helpful, others harmful

Recombination

a type of genetic transfer when one bacterium donates DNA to another bacterium (a type of genetic transfer) end result is a new strain different from both the donor and the original recipients

Exception to Frameshift Mutation

addition or deletion of bases in multiples of three does not disturb the reading frame

Role of Restriction endonucleases in bioengineering

allows biotechnologists to cleave DNA at desired sites, specifically at palindromes

Gel Electrophoresis

allows for DNA analysis; produces a readable pattern of DNA fragments - samples are placed in compartments in a soft agar gel and subjected to an electrical current - phosphate groups have a negative charge, which causes DNA to move toward the positive pole in the gel - larger fragments migrate more slowly; smaller fragments migrate more quickly - position of fragments are determined by staining the gel - creates a genetic fingerprint

Silent Mutation

alters the base but does not change the amino acid

Gram Positive Conjugation (key difference from Gram -)

an opening is created between two adjacent cells (rather than attachment to surface w/ pilus) - replicated DNA passes across from one cell to another

Mutation

any change to nucleotide sequence (and thus DNA) of a genome - most identifiable when genotypic change leads to phenotypic change - involves loss, addition, and/or rearrangement of base pairs

Recombinant organism

any organism that contains genes that originated in another organism

PCR: Thermal Cycler

automatically performs the cyclic temperature changes required for PCR

Significance of Resistance (R) Plasmids/Factors in Conjugation

bear genes for: - resistance to antibiotics/drugs, and heavy metals (Ni and Hg) - synthesis of virulence factors (toxins, enzymes, adhesion molecules)

Nonsense Mutation

changes a normal codon into a stop codon - almost always results in a nonfunctional protein

Genomic Library

collections of DNA clones that represent the entire genome of numerous organisms!

restriction fragment length polymorphisms (RFLPs)

differences in the cutting patterns of specific restriction endonucleases

restriction endonucleases

enzymes capable of recognizing foreign DNA and breaking the phosphodiester bonds between adjacent nucleotides on both strands of DNA; protects bacteria against incompatible DNA of bacteriophages

Cloning Vector: Bacteriophages

have the natural ability to inject DNA into bacterial hosts through transduction* - Vectors typically contain a gene that confers drug resistance to their cloning host - cells can be grown on drug containing media - only those cells that harbor a plasmid will be selected for growth

Cloning

involves removal of a selected gene from an animal, plant, or microorganism and propagated in a host microorganism - donor gene must be excised by restriction endonucleases and isolated - gene must be inserted into a vector (usually a plasmid or a virus) - vector inserts the gene into the cloning host - cloning host is usually a bacterium or yeast which can translate the gene into the desired protein

Missense Mutation

leads to a different amino acid. Can lead to: - a faulty, nonfunctional, or less functional protein - a protein that functions in a different manner - no significant alteration in protein function

Complementary DNA (cDNA)

made from mRNA, tRNA, and rRNA! - used to synthesize eukaryotic genes from mRNA!transcripts and is free from introns!

Conjugation DEFN

mode of genetic exchange in which a plasmid or other genetic material is transferred by a donor to a recipient cell (related species) via a direct connection/bridge *involves alive donor and recipient cells

Lethal Mutations

mutations that have a harmful effect and lead to dysfunction or death of the cell!

Ligase

necessary to seal the sticky ends together - used in the final splicing of genes into plasmids and chromosomes

Transformation DEFN

nonspecific (not necessarily same species) acceptance by a bacterial cell of small fragments of DNA from surrounding environment (from lysed bacterial cells) *DNA from lysed cell retains its sequence *involves alive, competent recipient cell

Back Mutation

occurs when a gene that has undergone mutation reverses (mutates back) to its original base composition

Frameshift Mutation

occurs when one or more bases are inserted or deleted from a DNA strand - reading frame of mRNA has been changed - nearly always results in a nonfunctional protein - every amino acid after the mutation is different from what was coded for in the original DNA

Difference between plasmids and chromosomal fragments

plasmids have own origin of replication and are stably replicated and inherited; chromosomal fragments must integrate themselves into bacterial chromosome in order to be replicated and passed on

Horizontal Gene transfer typically involves pieces of DNA in what forms? (2 possibilities*)

plasmids or chromosomal fragments

Transduction DEFN

process by which DNA is carried from one bacterium to another bacterium via a bacteriophage (bacterial virus) *donor is lysed cell, recipient is alive cell of the same species

Neutral Mutations

produce neither adverse or helpful changes

Results of Recombination

provide genes for 1) resistance to drugs and metabolic poisons 2) new nutritional and metabolic capabilities 3) increased virulence and adaptation to the environment

palindromes

sequences of DNA that are identical when read from the 5' to 3' or from 3' to 5'

Transfection

similar process to transformation (bacteria), but carried out in eukaryotic cells to form genetically modified yeasts, plants, and mice; proposed as future technique for curing genetic disease

Point Mutations

small mutations that affect only a single base on a gene *specifically an addition, deletion, or substitution of a single base

Plasmids

small, circular pieces of non-essential DNA that contain their own origin of replication - can replicate independently of the bacterial chromosome - found in many bacteria - contain genes that confer useful traits, such as antibiotic resistance

E.Coli High frequency recombination Details

the plasmid of the F+ cell (donor) becomes integrated into the F+ cell chromosome - when chromosome is replicated, begins to transfer to the recipient cell - partial chromosomal genes get transferred to the recipient before bridge/pilus is broken - plasmid genes may or may not be transferred

Horizontal Gene Transfer

transfer of DNA that results in organisms acquiring new genes that did not come directly come from parent organisms (which would be termed vertical transfer)

Reverse Transcriptase

used by retroviruses to synthesize DNA from RNA, DNA then used to make cDNA

Beneficial Mutation

very rare; provide the cell with a useful change in structure or physiology