micro #9 genetics

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micro #9 genetics

Genetics

1. Genetics: the study of the inheritance ( heredity_) of living things
a. Transmission (passing on) of traits from parent to offspring
b. Expression and variation of those traits
c. The structure and function of the genetic material
d. How this material changes
2. Takes place on several levels: organismal, chromosomal, molecular

Nature of genetic material

1. Must be able to self-replicate
2. Must be accurately _duplicated_ and separated from each daughter cell
Note: plasmids copy themselves

The Levels of Structure and Function of the Genome

1. Genome = sum of all genetic material of a cell (in bacteria on single chromosome)
2. Chromosome
3. Gene = coding for protein or regulating molecules

chromosome definition

A discrete cellular structure composed of a neatly packed DNA molecule (contained w/in nucleus)

Eukaryotic chromosomes

1. DNA molecule tightly wound around _histones (like a yo-yo___ proteins
2. Located in the nucleus
3. Vary in number from a few to hundreds (ex. ferns)
4. Can occur in _pairs__ (diploid) or singles (haploid ex. gametes_)
5. Appear linear

Bacterial chromosomes

1. Condensed and secured by means of histone-like proteins
2. Single, _circular__ chromosome. copied quick b/c of this

Gene

1. A certain segment of DNA that contains the necessary code to make a protein or _RNA__ molecule
2. Structural genes: code for proteins will be made
3. Regulatory genes: control gene _expression___
4. Sum of all types of genes is an organisms' genotype (ex. Tt, tt)
5. The expression of the genotype creates traits- the (_phenotype___
6. All organisms contain more genes in their genotype than are manifested as a phenotype at a given time

Structural genes

code for proteins which will be made

Regulatory genes

control gene expression______

Genotype

Sum of all types of genes is an organisms' genotype

Size and packaging of genomes

1. Vary greatly in size
a. Smallest viruses- 4 or 5 genes (don't replicate, reproduce - all done w/in host)
b. Escherichia coli- 4,288 genes
c. Human cell- 20,000 to 40,000 genes. At one point it was thought there was 100,000 genes because of complexity
2. The stretched-out DNA can be 1,000 times or more longer than the cell

James Watson and Francis Crick

1. 1953: James Watson and Francis Crick
a. Discovered structure DNA is a gigantic molecule (with the help of x-ray crystallography by _Rosalin Franklin___)
b. A type of nucleic acids
c. With two strands combined into a double helix

General structure of DNA

Basic unit: nucleotide
3 components (RNA vs. DNA):
DNA = phosphate & deoxyribose (sugars), nitrogenous base (A,T,C,G)
RNA = phosphate & ribose (sugars), nitrogenous base (A,U,C,G)

Nucleotides

1. Covalently bond to form a sugar-phosphate linkage- makes up the _backbone_ of each strand
2. Each sugar attaches to two phosphates
3. One bond is to the 5' carbon on deoxyribose
4. The other is to the 3' carbon

Nitrogenous Bases

1. Purines and pyrimidines (double vs. single)
2. Attach by covalent bonds at the 1' position of the sugar
3. Span the center of the molecule and pair with complementary__ bases from the other strands (pyrimidines always w/purines)
4. The paired bases are joined by hydrogen bonds
a. Easily broken
b. Allow the molecule to be "_unzipped___"
Adenine always pairs with _thymine_ (double H bond)
_guanine_ always pairs with cytosine (triple H bond)

_Antiparallel_ Arrangment

1. One side of the helix runs in the opposite direction of the other
2. One helix runs from 5' to 3' direction
3. The other runs from 3' to 5'

The Significance of DNA Structure

1. Arrangement of nitrogenous bases
a. Maintains the code during reproduction (conservative replication of DNA)
b. Provides variety

DNA Replication: Preserving the Code and Passing it On

1. The process of the genetic code duplicated and passed on to each offspring
2. Must be completed during a single generation time
3. Generated _5' to _3'

The Overall Replication Process

1. Requires the actions of 30 different _enzymes__
2. Functions include:
a. Separate the strands & keeping separated
b. Copy its template
c. Produce two new daughter molecules

_Semiconservative__ Replication

1. Each daughter molecule is identical to the parent in composition, but only one strand is completely new
2. The parent DNA molecule uncoils
3. The __hydrogen_ bonds between the base pairs are unzipped (helicase)
a. Separates the two strands
b. Exposes the nucleotide sequence of each strand to serve as templates
4. Two new strands are synthesized by attachment of the correct complementary nucleotides to each single-stranded template (never fully separated)

Origin of replication

1. Short sequence
2. Rich in A and T
3. Held together by only two H bonds rather than three
4. Less energy is required to separate the two strands

_Helicases__ bind to the DNA at the origin

1. Untwist the helix
2. Break the hydrogen bonds
3. Results in two separate strands

DNA Polymerase III

Main enzyme
1. Synthesizes a new daughter strand using the parental strand as a template
2. The process depends on several other enzymes as well, but key points about DNA polymerase III:
a. Nucleotides that need to be read by DNA polymerase III are buried in the double helix- so the DNA must first be unwound and the two strands separated
b. DNA polymerase III is unable to begin synthesizing a chain of nucleotides but can only continue to add nucleotides to an already existing chain. Can start with help of _RNA polymerase/primer__ (add to 3' end, read 3' to 5')
c. DNA polymerase III can only add nucleotides in one direction, so a new strand is always synthesized from 5' to 3'

Elongation and Termination of the Daughter Molecules

1. As replication proceeds, the newly produced double strand loops down
2. DNA polymerase I removes RNA primers and replaces them with DNA
3. When the forks come full circle and meet, _ligases__ move along the lagging strand
a. Begin initial linking of the fragments
b. Complete synthesis and separation of the two circular daughter molecules

..

1. Occasionally an incorrect base is added to the growing chain
2. Most are corrected
3. If not corrected, result in mutations
4. DNA polymerase III can detect incorrect, unmatching bases, excise them, and replace them with the correct base
5. DNA polymerase I can also _proof read & detect errors__ and repair the errors

Transcription and Translation
Central dogma

1. Genetic information flows from DNA to RNA to protein
a. The master code of DNA is used to synthesize an RNA molecule (process known as _transcription__)
b. The information in the RNA is used to produce proteins (_translation nucleic acid to AA_)
c. Exceptions: RNA viruses and retroviruses
2. Recently shown to be incomplete
a. In addition to the RNA that produces protein, other RNAs are used to regulate gene function
b. Many of the genetic _malfunction_ that cause human disease are found in these regulatory RNA segments

The Gene-Protein Connection

1. The Triplet Code and the Relationship to Proteins
a. Three consecutive bases on the DNA strand- called triplets
b. A gene differs from another in its composition of triplets (call for certain AA)
c. Each triplet represents a code for a particular amino acid
d. When the triplet code is transcribed and translated, it dictates the type and order of amino acids (AA can be polar or nonpolar, charged or uncharged) in a polypeptide chain
2. A protein's _primary__ structure determines its characteristic shape and function
3. Proteins ultimately determine phenotype
4. DNA is mainly a blueprint that tells the cell which kinds of proteins and RNAs to make and how to make them

The Major Participants in Transcription and Translation

1. Number of components participate, but most prominent:
a. mRNA - "cookbook" what goes in & how its made
b. tRNA
c. regulatory RNAs (at DNA level - what gets transcribed next)
d. Ribosomes (containing [32]_rRNA__)
e. several types of enzymes
f. storehouse of raw materials - includes sugars, AA nitrogen base
2. RNAs: Tools in the Cell's Assembly Line
a. RNA differs from DNA
i. Single stranded molecule
ii. Contains uracil instead of thymine
iii. The sugar is ribose
b. Many functional types, from small regulatory pieces to large structural ones
c. Only (33)_mRNA____is translated into a protein molecule

RNAs: Tools in the Cell's Assembly Line

1. RNA differs from DNA
a. Single stranded molecule
b. Contains uracil instead of thymine
c. The sugar is ribose
2. Many functional types, from small regulatory pieces to large structural ones
3. Only (33)_mRNA__is translated into a protein molecule

Messenger RNA: Carrying DNA's Message

copied 5' to 3'
1. A transcript of a structural gene or genes in the DNA
2. Synthesized by a process similar to synthesis of the leading strand during DNA replication
3. The message of this transcribed strand is later read as a series of triplets (_codons_)

Transfer RNA: The Key to Translation

tRNA = "cloverleaf"
1. Also a copy of a specific region of DNA
2. It is uniform in length (75-95 nucleotides long)
3. Contains sequences of bases that form hydrogen bonds with complementary sections of the same tRNA strand (folds up on itself)
4. At these points the molecule bends back upon itself into several hairpin loops, giving the molecule a cloverleaf structure that then folds into a complex, 3-D helix
5. Bottom loop of the cloverleaf exposes a triplet (the anticodon_) that designates the specificity of the tRNA and complements mRNA's codons
6. At the opposite end of the molecule (anticodon) is a binding site for the amino acid that is specific for that anticodon
7. For each of the 20 amino acids there is at least one specialized type of tRNA to carry it

The Ribosome: A Mobile Molecular Factory for Translation

1. The prokaryotic (70S) ribosome composed of tightly packed rRNA and protein (a lot of enzymes)
2. The rRNA component is a long polynucleotide molecule (folds up on itself)
3. The interactions of proteins and rRNA create the two _subunits__ of the ribosome that engage in final translation of the genetic code

Translation: The Second Stage of Gene Expression

1. All of the elements needed to synthesize a protein are brought together on the _ribosome__
2. Five stages:
1. Initiation - start codon recognized by tRNA, small subunit, large subunit
2. Elongation - more bead on string
3. Termination - stop codon
4. Protein folding - begins as soon as AA are added to chain based on polar/nonpolar
5. Protein processing - doesn't need anything added ex. sugar or lipo
Steps 1-3 are translation

Initiation of Translation

1. mRNA molecule leaves DNA transcription site (not true for all prokaryotes)
2. Is transported to ribosomes in the cytoplasm
3. Ribosomal subunits are specifically adapted to assembling and forming sites to hold the mRNA and tRNAs
4. Prokaryotic ribosomes
a. 70s size
i. 50s subunit
ii. 30s subunit
5. Eukaryotic ribosomes
b. 80s size
i. 60s subunit
ii. 40s subunit

Prokaryotic ribosomes

a. 70s size
i. 50s subunit
ii. 30s subunit

Eukaryotic ribosomes

b. 80s size
i. 60s subunit
ii. 40s subunit

...

1. The small subunit binds to the _5'___ end of the mRNA (starts at 5' end) read & synthesized 5' to 3'
2. Large subunit supplies enzymes for making peptide bonds on the protein
3. The ribosome scans the mRNA by moving in the 5' to 3' direction along the mRNA
4. The first codon is called the _start_ codon (AUG (methionine) but can rarely be GUG)
5. With the mRNA message in place on the ribosome, the tRNAs the enter with their amino acids
a. The complementary tRNA meets with the mRNA code
b. Guided by the two sites on the large subunit called the P site and the A site
c. The E site is where used tRNAs are released

The Message in Messenger RNA

1. The three base mRNA _codon_ codes for a specific amino acids

2. _Redundancy_ of the genetic code: a particular amino acid can be coded for by more than a single codon (helps prevent mutation - still get correct AA
3. Wobble-effect: (44) only 1st two nucleotides are required to encode the correct AA & the 3rd nucleotide doesn't change its sense. prevents mutation
DNA made 5' to 3'

The Termination of Protein Synthesis

1. Brought about by the presence of a termination codon: Examples (45) UAA, UAG & UGA

2. Often called _nonsense or stop_ codons
*Because they do not code for a tRNA
3. When reached, a special enzyme (termination factor) breaks the bond between the final tRNA and the finished polypeptide chain, releasing it from the ribosome

Modifications to Proteins

some protein need modifying
1. Before it is released from the ribosome it starts to fold upon itself to achieve its biologically active _tertiary__ conformation

2. _Postransitional__ modifications may be necessary (after translation occurs)
a. Starting amino acid may be clipped off
b. Cofactors added (examples) metal ions (iron, manganese, zinc) or organic compound
c. Join with other proteins to form quaternary levels of structure

Eukaryotic Transcription and Translation: Similar Yet Different

1. Start codon is also AUG, but it codes for a different form of _methionine_______
2. Eukaryotic mRNAs code for just one protein (bacteria can have more than one)
3. Why can't eukaryotic transcription and translation be simultaneous? b/c DNA is in nucleus & has to be passed thru pores to the ribosomes, modified before.

4. Modified mRNA in eukaryotes must pass through pores in the nuclear membrane and be carried to the ribosomes in the cytoplasm for translation
*transcript = in nucleus
* translation = in cytoplasm

Introns

sequences of bases that do not code for protein, not used

__Exons____-

coding regions that will be translated into protein
a. Called a split gene- requires further processing before translation
b.Transcription of the entire gene with both exons and introns occurs first, producing a pre-mRNA (pg 264)

Poly-A tail:

A series of _series of adenines_ is added to the mRNA molecule (protects it and directs it out of the nucleus)

Guanine cap

Guanine cap = helps protect from highly enzymatic cytoplasm
A spliceosome recognizes the exon-intron junctions and enzymatically cuts through them
The exons are joined end to end
Some introns do code for cell substances (in humans, introns represent 98% of the DNA)

The Genetics of Animal Viruses

1. Diverse
2. Some- nucleic acid is linear; others, circular
3. Most exist in a single molecule, but in a few it is in several
4. Most contain dsDNA or ssRNA, but other patterns exist. Why? the host cell have enzymes to work w/ds DNA & ssRNA
5. In all cases:
a. Viral nucleic acid penetrates the cell
b. The nucleic acid is introduced into the host's gene-processing machinery
c. The virus instructs the host's machinery to synthesize large numbers of new virus particles
d. Viral mRNA is translated into viral proteins on host cell ribosomes using host tRNA

Genetic Regulation of Protein Synthesis and Metabolism

1. Control mechanisms ensure that genes are active only when their products are required_______________
a. Enzymes are produced as they are needed
b. Prevents the waste of energy and materials
c. Antisense RNAs, micro RNAs, and riboswitches provide regulation in prokaryotes and eukaryotes

2. Prokaryotes organize collections of genes into _operons__ (structural operons when needed)
a. Coordinated set of genes regulated as a single unit
b. Either inducible or repressible
i. _Repressible_ -contain genes coding for anabolic enzymes; several genes in a series are turned off by the product synthesized by the enzyme (turn off operon)
ii. _Inducible_ -the operon is turned on by the substrate of the enzyme for which the structural genes code

The Lactose Operon: A Model for Inducible Gene Regulation in Bacteria

1. Best understood cell system for explaining control through genetic induction
2. Lactose (lac) operon (code for protein that breaks down lactose)
3. Regulates lactose metabolism in Escherichia coli
4. Three important features:
a. The regulator (a gene that codes for a protein capable of repressing the operon [a repressor]) - 1st part of operon
b. The control locus - where regulator works
i. Promoter- recognized by RNA polymerase
ii. Operator- a sequence that acts as an on/off switch for transcription
iii. The structural locus, made up of three genes each coding for a different enzyme needed to catabolize lactose

Sites on ribosomes (EPA)

E = exit
P = petidyl site
A = acceptor site

How many strands used to make mRNA

one

codons on

mRNA

triplets on

DNA

anticodon on

tRNA

wobble effect

multiple codes for an AA is helpful for eliminating errors

transcript in

nucleus

translation in

cytoplasm

transcription & translation is effecient

only in prokaryotes.
possible b/c no nucleus, no modification
single transcript can have AA________?

Lac Operon: Regulatory Region

1. __CAP__ - turned on when glucose levels are low
2. P-lac - promoter region for lac operon
3. _Operator/O__- site where repressor protein will sit if lactose absent
4. Associated Molecules -
a. CAP protein and cAMP bind together at CAP binding site __bending___ the DNA
i. Allows RNA polymerase to bind
b. _Repressor__ protein - when lactose is absent it's presence halts synthesis of lactose catabolism enzymes
i. Allolactose binds to this protein allowing the O site to be switched on (b/c the repressor can't sit on the O site

Associated Molecules

a. CAP protein and cAMP bind together at CAP binding site _bending___ the DNA
i. Allows RNA polymerase to bind
b. _Repressor__ protein - when lactose is absent it's presence halts synthesis of lactose catabolism enzymes
i. Allolactose binds to this protein allowing the O site to be switched on (b/c the repressor can't sit on the O site)

A Repressible Operon

1. Normally the operon is in the "on" mode and will be turned "off" only when the nutrient is no longer required
2. The excess nutrient serves as a _co-repressor___ needed to block the action of the operon
3. Example, arg operon

Antibiotics that Affect Transcription and Translation

1. Some infection therapy is based on the concept that certain drugs react with DNA, RNA, or ribosomes and alter genetic expression
2. Based on the premise that growth of the infectious agent will be inhibited by blocking its protein-synthesizing machinery selectively
3. Drugs that inhibit protein synthesis exert their influence on transcription or translation
4. Antibiotics often target the ribosome- inhibiting ribosomal function and ultimately protein synthesis

Mutations: Changes in the Genetic Code

1. Genetic change is the driving force of evolution
2. Mutation: when phenotypic changes are due to changes in the _genotype__(can be good, bad or indifferent)
a. An alteration in the nitrogen base sequence of DNA

3. Wild type: a microorganism that exhibits a natural, non-mutated characteristic
4. Mutant strain: when a microorganism bears a mutation
a. Useful for tracking genetic events,
b. Unraveling genetic organization, and
c. Pinpointing genetic markers

Causes of Mutations (2)

1. _Spontaneous__ mutation: random change in the DNA arising from errors in replication
2. Induced mutation: results from exposure to known _mutagen (Ex._chemically = Ethidium bromide. Radiation = UV, Ionizing-gamma rays, x-rays)

Categories of Mutations: Point mutations (4)

1. Point mutations: involve addition, deletion, or substitution of single bases (one A,T,C or G)
a. _Missense__ mutation: any change in the code that leads to placement of a different amino acid (redundancy & wobble effect help to avoid this) sickle cell
b. Can create a faulty, nonfunctional protein
c. Can produce a protein that functions in a different manner
d. Can cause no significant alteration in protein function
2. Nonsense mutation: changes a normal codon into a _Stop__ codon (usually results in a nonfunctional protein)
3. __Silent_ mutation: alters a base but does not change the amino acid and thus has no effect
4. Back-mutation: when a gene that has undergone mutation reverses to its original base composition

Categories of Mutations: Frameshift mutations:

1. Frameshift mutations: mutations that occur when one or more bases are inserted into or deleted from a newly synthesized DNA strand
a. Changes the _reading frame (codon)_ of the mRNA (and changes everything following it)
b. Nearly always result in a nonfunctional protein

Repair of Mutations

1. Most ordinary DNA damage is resolved by enzymatic systems specialized for finding and fixing such defects (DNA & RNA polymerase)
2. DNA that has been damaged by UV radiation
a. Typically _thyamine_ dimers are formed-due to H bonds
b. Restored by photoactivation or light repair
c. DNA _photolyase_- light-sensitive enzyme
3. Excision repair
a. Excise mutations by a series of enzymes
b. Remove incorrect bases and add correct one

Repair of Mutations: DNA that has been damaged by UV radiation

a. Typically _thyamine________ dimers are formed
b. Restored by photoactivation or light repair
c. DNA _photolyase_________- light-sensitive enzyme

Repair of Mutations: Excision repair

a. Excise mutations by a series of enzymes
b. Remove incorrect bases and add correct one

Positive and Negative Effects of Mutations

1. Mutations are permanent and inheritable
2. Most are harmful but some provide adaptive advantages

DNA Recombination Events: Recombination

Recombination: when one bacterium donates DNA to another bacterium
1. The end result is a new strain different from both the donor and the original recipient
2. Bacterial plasmids and gene exchange

DNA Recombination Events: Recombination organism

Recombinant organism: Any organism that contains (and expresses - also uses it__) genes that originated in another organism

Transmission of Genetic Material in Bacteria

1. Usually involves small pieces of DNA (plasmids or chromosomal fragments)
2. Plasmids can replicate _independently___ of the bacterial chromosome
3. Chromosomal fragments must _integrate__ themselves into the bacterial chromosome in order to replicate
4. Three means of genetic recombination in bacteria (76):
a. conjugation = donor cell w/pilus (have genes)
b. transformation = transfer of naked DNA & requires no special vehicle
c. transduction = DNA transfer mediated thru the action of a bacteria virus

transformation

transfer of naked DNA & requires no special vehicle
ex. picking up the loose DNA from cells that lysed. Mice experiment.

transduction

DNA transfer mediated thru the action of a bacteria virus

Biomedical Importance of Conjugation

1. _Resistance__- (R) plasmids, or factors- bear genes for resisting antibiotics (can grow in its presence of those antibiotics)
2. Can confer multiple resistance to antibiotics to a strain of bacteria
3. R factors can also carry resistance to heavy metals or for synthesizing virulence factors
***Benefit of resistance to heavy metals? Inhibitory growth of bacteria
***Benefit of virulence factors? more pathogenic, cause more disease

Griffith demonstrated that DNA released from a killed cell can be acquired by a live cell

-happens randomly
1. Later studies supported this
2. Nonspecific acceptance by a bacterial cell- transformation
3. Facilitated by special DNA-binding proteins on the _cell wall
4. _Compotent__ cells- capable of accepting genetic material
5. Useful for certain types of recombinant DNA technology

Conjugation

donor cell w/pilus (have genes)

1. What must a cell be able to create to perform conjugation? (82) pili
2. Where is this normally coded? plasmid
3. What are the differences between strain I & II? (84)

Can you think of a reason why strain I may have grown on the LB/AMP/STR plates? (85)

Is a plasmid necessary from normal survival? (86)

Mutation

when phenotypic changes are due to changes in the _genotype__(can be good, bad or indifferent)
a. An alteration in the nitrogen base sequence of DNA

Wild type

a microorganism that exhibits a natural, non-mutated characteristic

Mutant strain

when a microorganism bears a mutation
a. Useful for tracking genetic events,
b. Unraveling genetic organization, and
c. Pinpointing genetic markers

Categories of Mutations: Point mutations
Missense

a. _Missense__ mutation: any change in the code that leads to placement of a different amino acid (redundancy & wobble effect help to avoid this)
b. Can create a faulty, nonfunctional protein
c. Can produce a protein that functions in a different manner
d. Can cause no significant alteration in protein function

Categories of Mutations: Point mutations
Nonsense

Nonsense mutation: changes a normal codon into a _Stop__ codon (usually results in a nonfunctional protein)

Categories of Mutations: Point mutations
Silent

__Silent_ mutation: alters a base but does not change the amino acid and thus has no effect

Categories of Mutations: Point mutations
Back-mutation

Back-mutation: when a gene that has undergone mutation reverses to its original base composition

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