BACTERIAL GENETICS

MICROBIAL GENETICS; MARKER GROWTH

CONDITIONS OF SELECTION; AB resistance ; genes let grow in presence or absence of drug

Expression of genes is phenotype

RESTRICTION ENZYMES; Cleave DNA at specific sites to form DNA restriction fragments

PLASMIDS; small genetic elements capable of independent replication in bacteria & yeasts.

Introduction of these rest.fragments in plasmids leads to their amplification [also by PCR]

DNA,RNADNA; double stranded; double helix with

bases in center determine genetics. Each helical turn has a major/minor groove. Major groove exposed more so binds proteins regulating gene expression

anti parallel 5’to 3’, 3’to5’

complementary base A-T;C-G

Hydrogen bonds in centre

Template strand: coding strand

NUCLEOTIDE; 4 BASES +PHOSPHO 2’DEOXYRIBOSE

Length of DNA; thousands base pairs, kilobase pairs[kbp]

Viris single DNA 5 kbp; E.coli 4639 kbp 1mm length

Coiling ,super-coiling of DNA

RNA; single strand

URACIL INSTEAD OF THYMINE; A-U; C-G

mRNA; communicate gene seq.of DNA as mRNA to ribosomes,

RNARibosomes; ribosomal RNA [rRNA]+ proteins

tRNA ; translate mRNA to protein molecule

SIZE; tRNA few 100 bases ribosomal RNA ;3 types 120 ,1540,2900 bases & many proteins

STABILITY; rRNA, tRNA : 95% of total RNA

mRNA gene expression alters with demand with rapid metabolic turnover

sRNA regulators bind near 5’ end of mRNA, prevent ribo,translation

GENEUNIT OF HEREDITY

SEGMENT of DNA,whose nucleotide sequence carries the information of a biochemical/physiological function

Genes identified by PHENOTYPE;collective structural & physiological properties of cell or organism eg eye colour,drug resistance

GENOTYPE is chemical basis in DNA;alteration of sequence within a gene or organisations of genes

Eukaryotic genomeGenome; totality of genetic information in organism.

Carried on 2 or more LINEAR CHROMOSOMES in a nucleus surrounded by NM

Diploid cells have 2 homologous copies of each chromosome; evolutionarily divergent

Mutations, genetic changes are not detected in diploid cells due to compensation of homologue.

Expressed in haploid cells with single copy of genes

Gene expressionRecessive genes ; no phenotypic expression bec of homologue

Dominant genes; overrides homologue & expresses

Mitochondria/chloroplasts; circular mol. DNA few genes encoding organelle functionmost of which is by chromosomal genes.

PLASMIDS; small 2um circular DNA 6.3 kbp Independent replication,found in yeasts[euk] &prok. can be genetically manip.& introd. In cells

Repetitive DNAEUKARYOTE; in extragenic regions, non coding

Large quantities

PROKARYOTES;SSR; excessive length polymorphism

SSR; short-seq.repeats

STRs; short tandemly repeat sequences

Several to thousands of dispersed copies

INTRONS; intervening seq.of DNA not transcribed on mRNA.

PROKARYOTIC GENOME; Haploid1.GENES carried on chromosomes,for growth

Single circular genome;580---5220kbp

Brucella,Burkholderia, have 2 circular DNA mol.

2.Genes on plasmids; spread of drug R

Several…100kbp

REPLICONS: DNA circles [1&2] carring genetic information for self replication

TRANSPOSONS; no self replication

Genetic elements; several kbp

Contain information for transfer from one locus to other

In migration cause insertion mutations esp. short transposons 750…2000bp called insertion elements or insertion seq IS elements

All bacteria have charcteristic ISE

PLASMIDS also have ISE; important for Hfr strains

Complex transposons, have genes for special function as AB resistance flanked by ISE

Physically attached replicon; not independantly ;copies inserted in same or diff.replicons randomly. If plasmid insertion can be widely disseminated

VIRAL GENOME PRASITES AT GENETIC LEVEL; lytic, temperate phages

survive but cant grow without host

Debilitates/kills host ,lives on its energy, uses macromol.

Bacteriophages; viruses of prok. 5000 in 140 bact.genera

NA;DNA ds common; others RNA ss, ds; ss DNA

Coat; protein, lipid

REPLICATION: ds DNA linear; becomes circular at cohesive ends, complementary tails that hybradize

Ligation; phosphodiester bonds form at tails

Replicates

Linear DNA formed;cleaved & packaged inside head

Ss DNA of filamentous phages is converted to circular double stranded replicative form.

One strand is used as a template for ssDNA continuously a rolling circles. Ss DNA is cleaved,packaged with protein for extrusion

Ss RNA PHAGESSmallest extracellular particles with information for their own replication

RNA phage MS2, has 4000 nucleotides, 3 genes act as mRNA following infection;

1,coat protein

2.RNA polymerase ssRNA; ss formed from replicas

Template phagesProphage stage;

1 plasmid-like existance

2.host chromosome integration at int locus shared homology site

3.many sites of insertion like transposons

Repressed genes ; for lytic/vegetative replication

Immunity against similar phages

Derepression; triggered by mol.reaction/ uv light vegetative burst ,lysis esp in actively dividing cells

Pathgenicity islands clusters of genes in DNA possesing specific determinants of pathogenecity

Large; at least 200kbp

code virulence to invade higher organisms as adhesins,invasins,toxins

Diff. G:C content than rest of genome

Linked to tRNA genes flanked by direct repeats

Prok genetic transferWidespread, genetic diversity

Small fragment transferred to recipient

Replication of recombinent

1.integration of DNA in replicon

2. independent replicon

Restriction to gene transfer

Retriction enzymes…endonucleases d/d self DNA from nonself by res gene

enzymes hydolyze DNA at specific sites with DNA seq from 4---13 bases

This specficty of fragmentation is basis of genetic engineering

Bacteria recognize sites through enzymes &modify hem by methylation of adnine/cytosine by

Type 1 system; combined single multisubunit protein

Type 11; sparate endonucleases & methylases

plasmidsWide hosr range drug resistance

Narrow host range

Coexistance of plasmids in bacteria

Compatable

Incompatable; one lost at higher rateon bacterial cell division

Mechanism of recombination

DNA replicates

No replication,then find recipient DNA

RECOMBINATION

HOMOLOGOUS;close similarity in donor,recepient common ancestrol genes. Rec gene dysfunction can give rise to bacteria that maintain closely related genes

NON HOMOLOGOUS; enzyme-catalyzed recomb. between dissimilar

Prokaryote BasicsThe largest and most obvious division of living

organisms is into prokaryotes vs. eukaryotes.

Eukaryotes are defined as having their genetic material enclosed in a membrane-bound nucleus, separate from the cytoplasm. In addition, eukaryotes have other membrane-bound organelles such as mitochondria, lysosomes, and endoplasmic reticulum. almost all multicellular organisms are eukaryotes.

In contrast, the genome of prokaryotes is not in a separate compartment: it is located in the cytoplasm (although sometimes confined to a particular region called a “nucleoid”). Prokaryotes contain no membrane-bound organelles; their only membrane is the membrane that separates the cell form the outside world. Nearly all prokaryotes are unicellular.

Three Domains of Life

Prokaryote vs. Eukaryote Genetics

Prokaryotes are haploid, and they contain a single circular chromosome. In addition, prokaryotes often contain small circular DNA molecules called “plasmids”, that confer useful properties such as drug resistance. Only circular DNA molecules in prokaryotes can replicate.

In contrast, eukaryotes are often diploid, and eukaryotes have linear chromosomes, usually more than 1.

In eukaryotes, transcription of genes in RNA occurs in the nucleus, and translation of that RNA into protein occurs in the cytoplasm. The two processes are separated from each other.

In prokaryotes, translation is coupled to transcription: translation of the new RNA molecule starts before transcription is finished.

Bacterial CultureSurprisingly, many, perhaps even most, of the bacteria on Earth cannot be grown in the laboratory today.

Bacteria need a set of specific nutrients, the correct amount of oxygen, and a proper temperature to grow. The common gut bacterium Escherichia coli (E. coli) grows easily on partially digested extracts made from yeast and animal products, at 37 degrees in a normal atmosphere. These simple growth conditions have made E. coli a favorite lab organism, which is used as a model for other bacteria.

More Culture Bacteria are generally grown

in either of 2 ways: on solid media as individual colonies, or in liquid culture.

The nutrient broth for liquid culture allows rapid growth up to a maximum density. Liquid culture is easy and cheap.

Solid media use the same nutrient broth as liquid culture, solidifying it with agar. Agar a polysaccharide derived from seaweed that most bacteria can’t digest.

The purpose of growth on solid media is to isolate individual bacterial cells, then grow each cell up into a colony. This is the standard way to create a pure culture of bacteria. All cells of a colony are closely related to the original cell that started the colony, with only a small amount of genetic variation possible.

Solid media are also used to count the number of bacteria that were in a culture tube.

Bacterial Mutants Mutants in bacteria are mostly biochemical in

nature, because we can’t generally see the cells.

The most important mutants are auxotrophs. An auxotroph needs some nutrient that the wild type strain (prototroph) can make for itself. For example, a trp- auxotroph can’t make its own tryptophan (an amino acid). To grow trp- bacteria, you need to add tryptophan to the growth medium. Prototrophs are trp+; they don’t need any tryptophan supplied since they make their own.

Chemoauxotrophs are mutants that can’t use some nutrient (usually a sugar) that prototrophs can use as food. For example, lac- mutants can’t grow on lactose (milk sugar), but lac+ prototrophs can grow on lactose.

Resistance mutants confer resistance to some environmental toxin: drugs, heavy metals, bacteriophages, etc. For instance, AmpR causes bacteria to be resistant to ampicillin, a common antibiotic related to penicillin.

Auxotrophs and chemoauxotrophs are usually recessive; drug resistance mutants are usually dominant.

Replica Plating A common way to find bacterial mutants is replica plating,

which means making two identical copies of the colonies on a petri plate under different conditions.

For instance, if you were looking for trp- auxotrophs, one plate would contain added tryptophan and the other plate would not have any tryptophan in it.

Bacteria are first spread on the permissive plate, the plate that allows both mutants and wild type to grow, the plate containing tryptophan in this case. They are allowed to grow fOR a while, then a copy of the plate is made by pressing a piece of velvet

onto the surface of the plate, then moving it to a fresh plate with the restrictive condition (no tryptophan). The velvet transfers some cells from each colony to an identical position on the restrictive plate.

Colonies that grow on the permissive plate but not the restrictive plate are (probably) trp- auxotrophs, because they can only grow if tryptophan is supplied.

Replica Plating, pt. 2

BACTERIAL DNA unwound

Bacterial Sexual Processes

Eukaryotes have the processes of meiosis to reduce diploids to haploidy, and fertilization to return the cells to the diploid state.

Bacterial sexual processes are not so regular. However, they serve the same aim: to mix the genes from two different organisms together.

GENETIC TRANSFER/RECMBINATION

Exchange of genes between two DNA molecules to form new combinations of genes on a chromosome

Contribute to genetic diversity; evolution

Better than mutation as new function beneficial to microbe

Vertical gene transfer to offspring; plants, animals,

Horizontal: microbes via donor/recepient <1% of entire bacterial population; vertical transmission also in bacteria

No integration

Integration

GENETIC TRANSFERThe three bacterial sexual processes

1. Conjugation: direct transfer of DNA from one bacterial cell to another.

2. Transduction: use of a bacteriophage (bacterial virus) to transfer DNA between cells.

3. Transformation: naked DNA is taken up from the environment by bacterial cells.

TRANSFORMATIONTransfer of “naked” DNA between bacteria

Active process; needs specific proteins called “competence factors”

Fredrick Griffith in 1928 worked on 2 strains of S pneumoniae

Oswald Avery and associates 1944 proved the chemical material transferred was DNA.

Recombinant or hybrid; new cell transfers to descendants that are identical

DNA-mediated transformation

(transformation)

Discovered by Fredrick Griffith in 1928 while working with Streptococcus pneumoniae

Griffith realized S. pneumoniae existed in two formsEncapsulated, virulent form (smooth in appearance)Nonencapsulated, avirulent form (Rough in appearance)

Griffith hypothesized that injections with the smooth

strain could protect mice from pneumonia

Griffith injected mice with the two different strains

Griffith’s Results

TransformationNature: different genera of Niesseria, Haemophilus

Streptococcus, Staphylococcus, Acinetobacter

Best between closely related cells

DNA is a large molecule, passes only when cell wall in a physiological competent state.

Competence involves alterations in cell wall that make it permeable to large DNA molecule.

Occurs in late log and early stationary phase in nature

Dying cells rupture during the stationary and death phases. The chromosome breaks into small pieces and explodes through the ruptured cell wallRecipient cells absorb pieces of “naked” DNA

Enzymes cleave recipient DNA

The naked DNA is integrated into the recipient

cell’s DNA at that site

Naked DNA integrates at a homologous site on the recipient’s chromosome

TransformationRecombinant DNA work. remove DNA from cells, manipulate it in the test tube, then put it back into living cells.

In the case of E. coli, cells are made “competent” to be transformed by treatment with: calcium chloride ions

heat shock. E. coli cells in this condition readily pick up DNA from their surroundings and incorporate it into their genomes.

Conjugation

High frequency of recombination – Hfr strains

Conjugation

Conjugation is mediated by a plasmid R plasmids

F plasmids

Conjugation requires direct contact between cells

Cells must be of opposite mating types

Donor cells carry a plasmid that codes for fertility factor or “F

factor”This cell is designated F+

Recipient cell does not carry a plasmidThis cell is designated F-

CONJUGATION BY PLASMIDThe ability to conjugate is conferred by the F plasmid. can spontaneously be lost

A plasmid is a small circle of DNA that replicates independently of the chromosome. Bacterial cells that contain an F plasmid are called “F+”. Bacteria that don’t have an F plasmid are called “F-”.

F+ cells grow special tubes called “sex pilli” from their bodies. When an F+ cell bumps into an F- cell, the sex pilli hold them together, and a copy of the F plasmid is transferred from the F+ to the F-. Now both cells are F+.

When it exists as free plasmid, the F plasmid can only transfer it self; no use in genetics.

However if F plasmid can become incorporated into bacterial chromosome by a cross-over between F plasmid and the chromosome, the resulting bacterial cell is called “Hfr” ie High frequency of recombination”

Hfr bacteria conjugate like F+ do but they drag a copy of entire chromosome into F- cell

Hfr Conjugation

Interrupted Mating Chromosome transfer from

the Hfr into the F- is slow: it takes about 100 minutes to transfer the entire chromosome.

The conjugation process can be interrupted using a kitchen blender.

By interrupting the mating at various times you can determine the proportion of F- cells that have received a given marker.

This technique can be used to make a map of the circular E. coli chromosome.

Different Hfr StrainsThe F plasmid can incorporate into the chromosome in almost any position, and in either orientation. Note that the genes stay in fixed positions, but the genes enter the F- in different orders and times, based on where the F was incorporated in the Hfr.

Data are for initial time of entry of that gene into the F-.

gene Hfr 1 Hfr 2 Hfr 3

azi 8 29 88

ton 10 27 90

lac 17 20 3

gal 25 12 11

Intracellular Events in Conjugation

The piece of chromosome that enters the F- form the Hfr is linear. It is called the “exogenote”.

The F- cell’s own chromosome is circular. It is called the “endogenote”.

Only circular DNA replicates in bacteria, so genes on the exogenote must be transferred to the endogenote for the F- to propagate them.

This is done by recombination: 2 crossovers between homologous regions of the exogenote and the endogenote. In the absence of recombination, conjugation in ineffective: the exogenote enters the F-, but all the genes on it are lost as the bacterial cell reproduces.

TransductionTransduction is the process of moving bacterial DNA from one cell to another using a bacteriophage.

Bacteriophage or just “phage” are bacterial viruses.

They consist of a small piece of DNA inside a protein coat. The protein coat binds to the bacterial surface, then injects the phage DNA. The phage DNA then takes over the cell’s machinery and replicates many virus particles.

types

Two forms of transduction:

1. generalized: any piece of the bacterial genome can be transferred

2. specialized: only specific pieces of the chromosome can be transferred.

Transduction

General Phage Life Cycle

1. Phage attaches to the cell and injects its DNA.

2. Phage DNA replicates, and is transcribed into RNA, then translated into new phage proteins.

3. New phage particles are assembled.

4. Cell is lysed, releasing about 200 new phage particles.

Total time = about 15 minutes.

Generalized Transduction

Some phages, such as phage P1, break up the bacterial chromosome into small pieces, and then package it into some phage particles instead of their own DNA.

These chromosomal pieces are quite small: about 1 1/2 minutes of the E coli chromosome, which has a total length of 100 minutes.

A phage containing E coli DNA can infect a fresh host, because the binding to the cell surface and injection of DNA is caused by the phage proteins.

After infection by such a phage, the cell contains an exogenote (linear DNA injected by the phage) and an endogenote (circular DNA that is the host’s chromosome).

A double crossover event puts the exogenote’s genes onto the chromosome, allowing them to be propagated.

Transduction MappingOnly a small amount of chromosome, a few genes, can be transferred by transduction. The closer 2 genes are to each other, the more likely they are to be transduced by the same phage. Thus, “co-transduction frequency” is the key parameter used in mapping genes by transduction.

Transduction mapping is for fine-scale mapping only. Conjugation mapping is used for mapping the major features of the entire chromosome.

Mapping ExperimentImportant point: the closer 2 genes are to each

other, the higher the co-transduction frequency.

We are just trying to get the order of the genes here, not put actual distances on the map.

Expt: donor strain is aziR leu+ thr+. Phage P1 is grown on the donor strain, and then the resulting phage are mixed with the recipient strain: aziS leu- thr-. The bacteria that survive are then tested for various markers

1. Of the leu+ cells, 50% are aziR, and 2% are thr+. From this we can conclude that azi and leu are near each other, and that leu and thr are far apart.

But: what is the order: leu--azi--thr, or azi--leu--thr ?

Mapping Experiment, pt. 2

2. Do a second experiment to determine the order. Select the thr+ cells, then determine how many of them have the other 2 markers. 3% are also leu+ and 0% are also aziR.

By this we can see that thr is closer to leu than it is to azi, because thr and azi are so far apart that they are never co-transduced.

Thus the order must be thr--leu--azi.Note that the co-transduction frequency for thr and leu are slightly different for the 2 experiments: 2% and 3%. This is attributable to experimental error.

Larger ExperimentA few hints:

1. There are 3 experiments shown. In each, 1 gene is selected, and the frequencies of co-transduction with the other genes is shown.

2. start with 2 genes that are selected and that have a non-zero co-transduction frequency. Put them on the map.

3. Then locate the other genes relative to the first 2.

selected

co-transduced

freq selected

co-transduced

freq selected

co-transduced

freq

e a 0 f a 90 c a 74

e b 85 f b 2 c b 32

e c 29 f c 41 c d 0

e d 62 f d 0 c e 21

e f 0 f e 0 c f 39

Intro to Specialized Transduction

Some phages can transfer only particular genes to other bacteria.

Phage lambda (λ) has this property. To understand specialized transduction, we need to examine the phage lambda life cycle.

lambda has 2 distinct phases of its life cycle. The “lytic” phase is the same as we saw with the general phage life cycle: the phage infects the cell, makes more copies of itself, then lyses the cell to release the new phage.

Lysogenic PhaseThe “lysogenic”: the lambda phage binds to the bacterial cell and injects its DNA.

Once inside the cell, the lambda DNA circularizes, then incorporates into the bacterial chromosome by a crossover, similar to the conversion of an F plasmid into an Hfr.

Once incorporated into the chromosome, the lambda DNA becomes quiescent: its genes are not expressed and it remains a passive element on the chromosome, being replicated along with the rest of the chromosome. The lambda DNA in this condition is called the “prophage”.

reproducing itself, then lysing the cell.

After many generations of the cell, conditions might get harsh. For lambda, bad conditions are signaled when DNA damage occurs.

When the lambda prophage receives the DNA damage signal, it loops out and has a crossover, removing itself from the chromosome.

Then the lambda genes become active and it goes into the lytic phase,

More Lysogenic Phase

Specialized TransductionUnlike the F plasmid that can incorporate anywhere in the E coli genome, lambda can only incorporate into a specific site, called attλ.

The gal gene is on one side of attλ and the bio gene (biotin synthesis) is on the other side.

Sometimes when lambda come out of the chromosome at the end of the lysogenic phase, it crosses over at the wrong point. This is very similar to the production of an F’ from an Hfr.

When this happens, a piece of the E coli chromosome is incorporated into the lambda phage chromosome

These phage that carry an E coli gene in addition to the lambda genes are called “specialized transducing phages”. They can carry either the gal gene or the bio gene to other E coli.

Thus it is possible to quickly develop merodiploids (partial diploids) for any allele you like of gal or bio.

Note that this trick can’t be used with other genes, but only for genes that flank the attachment site for lambda or another lysogenic phage.

PLASMIDSGENETIC ELEMENTS; 1/5size of bacterial DNA

Additional mechanism of genetic exchange; selective advantage in an environment

Present in prokaryotes and rarely eukaryotes

Self replicating autonomously called Replicons;

horizontal transmission by conjugation, tran, trans

Used as vectors for molecular cloning recombining sequences; Gene therapy in humans

PLASMIDS

2 TYPES Integrated/Non

TYPES OF PLASMIDS1.F-PLASMID :

CONJUGATIVE PLASMID

WITH GENE FOR SEX PILUS

WITH GENE FOR TRANSFER TO OTHER CELL

2.DISSIMILATION PLASMID:

Code for enzymes that trigger catabolism of unusual sugars and hydrocarbons

PSEUDOMONASToluene

Camphor

Hydrocarbons of petrolium

Survival value in adverse conditions

USE: Cleanup of environmental wastes

3.Pathogenicity of Bacteria

Eg E coli: harmless commensal of large gut

Strains causing infant diarrhea & traveler’s diarrhea

Code for: 1 .toxin production

2. intestinal attachment

S aureus: Exfoliative toxin

Cl tetani: neurotoxin

B anthrax: toxin

Bacteriocins synthesis genes in plasmids

RESISTANCE FACTORS: R factors

Discovered in Japan in1950 in dysentry cases

Resistance to one or >anti-biotics

Resistance in normal flora too eg E coli

Spread of plasmid mediating transfer is called R factors

AMP & TETR R GENES

R FACTORSResistance to AB, Heavy metals, Cellular toxins

2 components:

R transfer factors: genes for plasmid replication and conjugation

R determinant: resistant genes

code for enzymes inactivating AB & toxins

Multiple R factors in a bacterium can combine giving new combinations of r determinants

AB ResistanceWidespread use in industry, agriculture, animal feed

Preferential selection of AB R bacteria

R bacteria grow and expand within same species

And other species eg Neisseria acquired pencillinase-producing plasmid from Streptococcus and Agrobacterium

Non conjugative plasmid can insert in conjugative plasmid or chromosome; or by transformation

TRANSPOSONSmall DNA segments; 700-4000 base pairs long.

Can ”Transpose” from one DNA region to another of wide host range; bacteria….humans

Discovered in 1950 in corn but now seen in all microorganisms by Barbra McClintok

They move within one chromosome from one site to another, or to another chromosome or to a plasmid.

Rare phenomenon like mutation at frequency of 10

-1-10-7; Role in evolution

Transpose mechanismDirectly: cut paste

Make copies: these transpose

Effects:

Interrupt the normal spelling of DNA

Interrupt protein formation by putting oFF or increase by putting ON

Gene mutation

Survival value: AB resistance, make new proteins

TYPESContain information of their own transposition

SIMPLEST: Insertion sequences contain a gene for enzyme transposase….catalyzes cutting and resealing of DNA

Recognition sites are short inverted repeat sequences that the enzyme recognizes as recombination sites between chromosome and transposon

Complex transposonsCarry genes other than transpositioning eg

Endotoxin gene

AB resistance gene

Plasmids as R factors are made of a collection of transposons

Function: natural mechanism of gene movement from one chromosome to other

From one organism to another via plasmids, viruses

MUTATIONSA change in base sequence of DNA

It may alter a product encoded by that gene

EFFECT:

Disadvantage: eg enzyme may be rendered inactive Lethal: may be lethal mutation

Beneficial: give enhanced activity to organism

TYPESSILENT: neutral ie no effect on activity of product encoded by the gene

Eg one nucleotide substitution in DNA for another at position 3 of mRNA codon

A nucleotide substitution may still encode for same aa or even change in aa may not bring a change

May not alter the structure, function of gene product or a minor alteration in nonfunctional part may occur

BASE SUBSTITUTION: point mutation

Single base at one point of DNA seq is substituted with a different base eg AT for GC, or GC for CG;

If protein is encoded mRNA will transcribe an incorrect base, hence incorrect aa translated

This is MISSENSE MUTATION

Effect: dramatic as in sickle cell disease. A missense change A to a T results in aa valine instead ofglutamic acid

Shape of Hb changes esp under low O2 , shape of RBC changes, movement of RBC in capillaries is impeded

A STOP (non sense) codon may be created in the middle of mRNA molecule; some base substitutions prevent creation of functional protein; Only a fragment is made.

A base substitution ending in a NONSENSE CODON is called a nonsense mutation

FRAMESHIFT MUTATIONFew nucleotide seq are added or deleted in DNA

Huntington’s chorea: many bases added to a gene.

This alteration shifts the “translational reading frame” ie the 3 by 3 nucleotide grouping read as CODONS by tRNAs during translation.

Eg deleting a nucleotide pair in the mid gene may change many aa downstream from site of original mutation. So long stretch of altered aa made resulting in inactive protein at site beyond mutation. usually a nonsense codon is encountered that terminates the translation.

MUTATIONSSpontaneous: mistake during DNA replication

Mutagens: chemical: household

radiations: X rays, UV light

physical

Bacteria: AB resistance, altered cell membrane, capsule are mutations

CHEMICALS1.Nitrous acid: Random base substitution

A does not pair T but C. so in progeny AT is replaced by CG

2.Nucleoside analog: structurally similar to bases but base pairing different

5 bromouracil, substitutes thymine and pairs cytosine

2 aminopurine (substitutes adenine but may pair with guanine

Such analogs when added to growing cells, they are incorporated in DNA, substitute bases AND MISPAIR. Passed onto daughter cells as mutations

Antiviral and anti-tumor drugs are nucleoside analogs

Frameshift mutagens: often potent carcinogens

Benzpyrene in smoke and soot causes

Aflatoxin made by Aspergillus flavus in peanuts

RADIATIONSX rays

Gamma rays

Ionize atoms; electrons pop out from shells, bombard more molecules to cause more damage resulting in reactive ions and free radicles (molecular fragments with unpaired electron)

Bind,damage DNA bases, erors in replication/repair

Physical breaks in backbone: covalent bonds broken

UV LIGHTNon-ionizing component of ordinary light

Mutagenic component is 260nm screened by ozone layer

Harmful covalent bonds made between based

Adjacent thymine dimers form which unrepaired can cause mutation.

REPAIR: Light repair enzymes

Nucleotide excision repair

Enzymes cut out distorted cross-limked thymines by opening wide gap; excision repair defect in xeroderma pigmentosa; inherited. UV light sensitivi

Fill gap by complimentary strand

Restore original base pair sequence

DNA ligase seals it

If error remains…..it is mutation

Sun tann: large no of thymine dimers in skin; cancers