Download Microbial Genetics Chromosomes Genes Related to Obesity in the

Chromosomes
•Chromosome: discrete cellular structure composed of a neatly packaged DNA molecule
Microbial Genetics
•Eukaryotic chromosomes
- DNA wound around histones
- located in the nucleus
- diploid (in pairs) or haploid (single)
- linear appearance
•Prokaryotic chromosomes
- DNA condensed into a packet by means of histone‐like proteins
- single, circular chromosome
Genes Related to Obesity in the Human Genome
Map of E. coli’s ~5000 Genes
• Notice it is single & circular
• Does E. coli have 1 or 2 alleles of each gene? How do you know?
• Humans were first thought to function with 100,000 genes and now the number has dropped to ~35,000 genes although this is still a hot topic in research
Genome
Genome: sum total of genetic material of an organism
-most of the genome exists in the form of chromosomes
-some appears as plasmids or in certain organelles of eukaryotes
-genome of cells composed entirely of DNA
E. coli cell disrupted to release its DNA molecule.
-genome of viruses can contain either DNA or RNA 1
Gene
Genetic Terms
• Genotype
• an organism’s genetic makeup; its entire complement of DNA
• A gene is a segment of DNA that contains the necessary code to make a protein or RNA molecule
•Three categories of genes
• structural genes: code for proteins
• genes that code for RNA machinery used in protein production
• regulatory genes: control gene expression
• Phenotype
• is the expression of the genes: the proteins of the cell and the properties they confer on the organism.
• Size, shape, color, environment
Dracunculus vulgaris
The DNA Code
H
N
N
N–H
G
H
H–N
O
N
• Nucleotide: basic unit of DNA structure
• phosphate
• deoxyribose sugar
• nitrogenous base
Nitrogenous Bases and Base Pairing
Hydrogen bond
H
C
• Pairing dictated by the formation of hydrogen bonds between bases
• Complementary Base Pairing– if sequence of one strand known, sequence of other strand inferred
• Try it: H
N
N
N–H
Sugar
O
3′ OH
H
P
D
5′
4′
D
1′
5′
P
2′
P
C
G
D
3′
P
O
A
D
O
P
O
P
O
C
D
P
P
O
T
D
D
G
O
D
O
P
• Nucleotides covalently bond to each other in a sugar‐phosphate linkage
P
C
G
D
D
P
P
T
D
A
D
O
P
O
D
P
O
C
G
O
D
O
P
P
P
T
A
D
D
P
5′
D
D
3′
H
N–H
N
N
N
O
CH3
H– N T
H
N
N
Sugar
TAC GTA ACG
5′
H
OH
H
O
Hydrogen bond
ATG CAT TGC
(a)
Nature of the Double Helix
- Antiparallel arrangement:
one side of the helix runs in the opposite direction of the other
- One side runs from 5’ to 3,’ and the other side runs 3’ to 5’
DNA Replication
DNA  DNA
- This is a significant factor in DNA synthesis and protein production
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DNA Replication
DNA Replication
• DNA replication involves unwinding a DNA double helix and using each strand as a template for a new, complementary strand
• DNA polymerase and over a dozen other enzymes and proteins are required to successfully replicate a single strand of DNA
• DNA replication is semi‐
conservative since each new chromosome will have one “old” and one “new” strand
• When does this occur?? • What is needed to replicate DNA:
1. Original DNA template 2. Nucleotides
• a pool of nucleotides is free floating in the cytoplasm 3. Enzymes
• DNA polymerase, ligase 4. Energy • ATP
DNA Replication: Prokaryotes
• Certain enzymes unwind the DNA.
• Then, DNA polymerase can read the parent strand and attach a complementary nucleotide to the new strand of DNA.
• Nucleotides are free in the cytoplasm.
Transcription
DNA  RNA
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DNA vs. RNA
– Contains ribose rather than deoxyribose
– RNA is single stranded
– There is no T in RNA. Instead it is a U:
• A:U in RNA
– Can assume secondary and tertiary levels of complexity, leading to specialized forms of RNA (tRNA and rRNA)
Transcription: RNA Synthesis
• What you need to synthesize RNA:
1. Original DNA template: • chromosome with a promoter site (DNA sequence indicating start site) and a terminator site
2. Nucleotides
• G, C, A, U Uracil is substituted for thymine
3. Enzymes
• RNA polymerase 4. Energy • ATP
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Transcription
• RNA polymerase: large, complex enzyme that directs the conversion of DNA into RNA
• Template strand: only one strand of DNA that contains meaningful instructions for synthesis of a functioning polypeptide
Transcription
Many types of RNA can be transcribed:
1. Messenger RNA (mRNA)
• RNA molecule that serves as a message of the protein to be produced
2. Transfer RNA(tRNA)
• Transfers amino acids to ribosome 3. Ribosomal RNA (rRNA)
• Forms the ribosome
4. Regulatory RNA
• micro RNAs, anti‐sense RNAs, riboswitches, small interfering RNAs
Transcription: Initiation
Transcription: Elongation
Direction of
transcription
Early mRNA
transcript
Nucleotide
pool
• RNA polymerase recognizes promoter region
• RNA polymerase begins its transcription at a special codon called the initiation codon
• As the DNA helix unwinds it moves down the DNA synthesizing RNA molecule • During elongation the mRNA is built, which proceeds
in the 5’ to 3’direction (you do not need to know the
direction of elongation for this class)
• The mRNA is assembled by the adding nucleotides
that are complementary to the DNA template.
• As elongation continues, the part of DNA already
transcribed is rewound into its original helical form.
Transcription: Termination
Practice Transcription
Elongation
• DNA: GCGGTACGCATTAAGCGCCC
Late mRNA
transcript
• RNA:
At termination the polymerases recognize
another code that signals the separation
and release of the mRNA strand,
or transcript.
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Translation
• Decoding the “language” of nucleotides and converting/translating that information into the “language” of proteins.
• The nucleic acid “language” is in the form of codons, groups of three mRNA nucleotides.
• The protein “language” is in the form of amino acids
Translation
RNA  Protien
Translation
tRNA
• Decoder molecule which serves as a link to translate the RNA language into protein language – One site of the tRNA has an anticodon which complements the codon of mRNA
– The other site of the tRNA has an amino acid attachment site corresponding to a specific amino acid as noted in the genetic code
• Translation occurs at the ribosome
• The green mRNA strand is “threaded” through the ribosome.
• The ribosome “reads” the mRNA strand codons with the help of the genetic code and tRNA
Translation and the “Genetic Code”
Translation and the “Genetic Code”
• Triplet code that specifies a given amino acid
• There is one start codon, AUG, that codes for the amino acid methionine.
• We use the “genetic code” (at right) to translate mRNA nucleotide sequence (codons) into amino acid sequence which make up proteins.
•
The “genetic code” is degenerate which allows for a certain amount of mutation. I.e. UUU and UUC both code for Phe
•
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There are 3 stop codons, UAA, UAG and UGA that signal the ribosome to stop translation and let go of the polypeptide chain (protein).
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Practice Translation
Practice Translation
• RNA:
• RNA:
CGCCAUGCGUAAUUCGCGGG
CGCCAUGCGUAAUUCGCGGG
1st Step: Find the start of the gene which is always indicated by AUG. Everything upstream from that can be ignored.
1st Step: Find the start of the gene which is always indicated by AUG. Everything upstream from that can be ignored.
Practice Translation
• RNA:
AUG/CGU/AAU/UCG/CGG/G
2nd Step: To make it easier to track the codons I separate each with a slash
Translation at the Molecular Level
• Ribosomes bind mRNA near the start codon (ex. AUG) • tRNA anticodon with attached amino acid binds to the start codon
Practice Translation
• RNA:
AUG/CGU/AAU/UCG/CGG/G
3rd Step: Use genetic code to translate mRNA message into amino acid language
Translation at the Molecular Level
• Ribosomes move to the next codon, allowing a new tRNA to bind and add another amino acid
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Translation at the Molecular Level
• Series of amino acids form peptide bonds Polyribosomal Complex
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-
-
A single mRNA is long enough to be fed through more than one ribosome
Permits the synthesis of hundreds of protein molecules from the same mRNA transcript
Would you see this in Eukaryotes?
Translation at the Molecular Level
• Stop codon terminates translation
Transcription and Translation in Eukaryotes and Prokaryotes
• Similar to prokaryotes except
– AUG encodes for a different form of methionine
– Transcription and translation are not simultaneous in eukaryotes
– Eukaryotes must splice out introns to achieve a mature mRNA strand ready to go to the ribosome. Gene Regulation
• Cells regulate genes in 3 major ways:
1. Feedback inhibition
– The end‐product inhibits the pathway (similar to a thermostat….when it reaches the desired temperature it turns off)
Operons
- Only found in bacteria
- Coordinated set of genes to make proteins that are needed at the same time
- all regulated as a single unit
- either inducible or repressible
2. Enzyme induction
– If a substrate is present, the enzyme for the substrate is induced.
3. Enzyme repression
a. If a nutrient is present, the enzyme to make it is repressed.
b. If a nutrient is absent, the enzyme to make it is turned on.
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lac Operon
• Most studied operon
• When lactose is absent
the repressor blocks RNA
Polymerase from binding
to the operator and
transcribing downstream
genes.
• When lactose is present it
binds to the repressor and
it falls off the operator
allowing RNA Polymerase
to bind.
• The downstream genes
are responsible for
digesting lactose and are
only on when lactose is
present.
Phase Variation
• Bacteria turn on or off a complement of genes that leads to obvious phenotypic changes
• New environment new phenotype!
• Most often traits affecting the bacterial cell surface
• Examples: - Neisseria gonorrhoeae: production of attachment fimbriae
- Streptococcus pneumoniae: production of a capsule
Types of Mutations
• Point Mutation
• put the cat out‐‐‐>puc the cat out
• put the cat out‐‐‐>put
• Frameshift (reading frame of mRNA shifts)
•
•
•
•
put the cat out‐‐‐>put hec ato ut
Deletion
Addition
Duplication
Using the lac Operon in
Genetic Research
• The LacZ gene was knocked into the Nkx2.2 gene to track
where Nkx2.2 is expressed in the mouse embryo
• You can also use the lac operon to control genes by
adding lactose to the system
Mutations
• A change in the sequence of DNA
• Possible effects of mutations
• No effect‐‐>no change in a.a. sequence • Good‐‐>new aa. Seq
– Increases variability in the gene pool, this is evolution!
• Bad‐‐>new aa. Seq
• Cancer is the product of a combination of bad mutations.
The Effects of a Point Mutation • When a base is substituted in DNA the mutation may have 2 effects:
– Changes the amino acid
– Does not change the amino acid
– Why doesn’t a mutation always change the amino acid sequence? 8
The Effects of Frameshift Mutations
• The addition, deletion or insertion of one or more nucleotides drastically changes the amino acid sequence.
Mutagen Examples • 5‐Bromouracil and acridine are 2 mutagen examples that can “insert” themselves in DNA and cause errors in DNA replication, transcription and translation. • Notice how similar in structure mutagens can be. There is just one change to thymine that can have dire consequences
What is the connection to cancer?
• Cancer is a genetic disease. It is the consequence of a change in DNA sequence.
• Carcinogen=substance that causes cancer
• Are mutagens also carcinogens?
• The Ames Test uses bacteria to identify possible carcinogens by looking for mutations to occur. Once a mutagen is identified, it is tested in animals to test if it is a carcinogen.
Mutation Rates
• Normal Mutation Rate‐ 1/1 million per gene
– Mutations are constantly occurring since our enzymes are not 100% perfect. • Mutagen‐ chemical or radiation that bring about mutations.
• Mutagen Mutation Rate= 1/1000‐1/100,000 per gene (10‐1000X the normal rate)
Thymine Dimers Caused by Radiation
• Radiation, such as X‐rays and UV rays, can cause dimers to form in DNA.
• Thymine dimers can interfere with DNA replication, transcription and translation.
Ames Test
The Ames test is used to screen environmental and dietary chemicals for mutagenicity and carcinogenicity without using animal studies.
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Genetic Recombination
• During meiosis of human gametes
• In bacteria, occurs when DNA is transferred between bacteria.
• Increases diversity in gene pool
• End result is a new strain different from both the donor and the original recipients
• Vertical gene transfer‐
Genetic Recombination
- Depends on the fact that bacteria have plasmids and are adept at interchanging genes
- Provide genes for resistance to drugs and metabolic poisons, new nutritional and metabolic capabilities, and increased virulence and adaptation to the environment
• Genes/DNA passed from an organism to its offspring
• Horizontal gene transfer‐
• Genes/DNA transferred between organisms
Plasmids
Self‐replicating circular pieces of DNA
1‐5% the size of bacterial chromosome
“mini‐chromosome”
Bacteria can store up many different plasmids for their use & can transfer these to other bacteria. • They can contain any gene that the bacteria don’t require but are useful to the survival of the bacteria. For example antibiotic resistance genes, toxin production, etc.
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•
•
•
Antibiotic Resistance (R) Plasmids
• Some plasmids can carry many antibiotic resistance genes.
• When bacteria collect many plasmids and these plasmids have many antibiotic resistance genes, a “superbug” may originate.
Conjugation
Three Types of Genetic Transfer in Bacteria
Conjugation
Transformation
Transduction
• A donor cell contains a F (fertility) plasmid making it F+.
• A conjugation pilus (genes for which are on the F+ plasmid) forms and the donor cell transfers a copy of the F plasmid to the recipient.
• Now, both cells have a F plasmid
• F+ plasmids can have other genes on them too, for example antibody resistance containing genes
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Transformation
Hfr Conjugation
• High frequency recombination (Hfr) donors contain the F factor in the chromosome
• Donor gives part of its chromosome to the recipient
• This transfers more genes to the recipient bacteria
• Very fast evolution for the recipient!
• Occurs when naked DNA fragments of one bacteria are close to another living cell. • Some bacteria have the ability to pick up naked DNA fragments and recombine the DNA into their own DNA
• The new recombinant cell now has some new DNA from the disintegrating cell. • The now transformed bacteria could have just picked up a new virulence factor or antibody resistance
Donor
Hfr cell
Partial copy
of donor
chromosome
Integration of
F factor into
chromosome
Pilus
Bridge
broken
Donated
genes
Griffith’s Classic Experiment to Prove Transformation
Mechanism of Transduction
• Virus mediated gene transfer
• The virus injects its genetic material into the bacteria
• The bacterial DNA is fragmented
Mechanism of Transduction
• Viral particles are produced by the bacteria
• When the cell lyses, the viral particles which have picked up DNA from the original bacterial cell now insert that DNA into a new cell.
• The new cell may or may not insert the new DNA sequence into its chromosome.
• Transduction can be a problem when the inserted DNA codes for an antibiotic resistance gene.
Transformation and Transduction in Research
Electroporation
A way to get the genes
you want to work with
into bacteria. Used in
all types of molecular
genetics research
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Transposons
• Transposons‐
• Small segments of DNA that can move (be transposed) from one region of a DNA molecule to another.
• “jumping genes”
– Involved in
• Changes in traits such as colony morphology, pigmentation, and antigenic characteristics
• Replacement of damaged DNA
• Intermicrobial transfer of drug resistance (in bacteria)
Genes & Evolution
• Genes are continually altered due to mutation, recombination, and transposition
• These changes increase genetic diversity of the gene pool and then natural selection acts on diverse populations to ensure survival in many different habitats.
• For pathogens that means they are more virulent!
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