genetics 192021222324

Gene mutations

molecular changes in the DNA sequence of a gene

transition

a change of a pyrimidine (C, T) to another pyrimidine (T, C) or a purine (A, G) to another purine (G, A)

transversion

a change of a pyrimidine (C, T) to a purine (A, G) or a purine (A, G) to a pyrimidine (C, T)

Chemically, transition mutations occur

more easily than transversion mutations

Silent mutations

those base substitutions that do not alter the amino acid sequence of the polypeptide due to the degeneracy of the genetic code

Missense mutations

those base substitutions in which an amino acid change occurs

Nonsense mutations

those base substitutions that change a normal codon to a stop codon

Frameshift mutations

involve the addition or deletion of a number of nucleotides that is not divisible by three

Deleterious mutations

decrease the chances of survival

Beneficial mutations

enhance the survival or reproductive success of an organism

Suppressor mutations

reverse the phenotypic effects of another mutation

In an intragenic suppressor mutant

the second mutant site is within the same gene as the first mutation

In an intergenic suppressor mutant

the second mutant site is in a different gene from the first mutation

Germ-line cells

Cells that give rise to gametes such as eggs and sperm

Somatic cells

All other cells

Lederberg bacteriophage T1 tonr experiment

Proved that spontaneous mutants could occur that provided selective advantages

Spontaneous mutations can arise by three types of chemical changes

1. Depurination
2. Deamination
3. Tautomeric shift

Depurination

the removal of a purine (adenine or guanine) from the DNA forming an apurinic site

Depurination of A or G can cause

both transition and transversion mutants

The deamination of cytosine

the removal of an amino group from the cytosine base; causes a C:G to T:A transition mutation

The deamination of 5-methylcytosine

causes a C:G to T:A transition mutation

the keto form

The common, stable form of thymine and guanine

an enol form

Rarely, T and G convert

the amino form

The common, stable form of adenine and cytosine

an imino form

Rarely, A and C can convert

Tautomeric shifts

cause transition mutations.

Oxidadtive stress

causes a transversion mutation

Deamination agents

cause transition mutations

Alkylating agents

cause transition mutations

Intercalating agents

cause frameshift mutations

Base analogues

cause transition mutations

UV can cause

the formation of cross-linked thymine dimers and transition mutations

The Ames test

uses a strain of Salmonella typhimurium that cannot synthesize the amino acid histidine. It has a point mutation in a gene involved in histidine biosynthesis that renders it non-functional.

A second mutation (that is a reversion)

may occur restoring the ability to synthesize histidine

The Ames test monitors

the rate at which this second mutation occurs

The covalent modifications of nucleotides can be reversed by three specific enzymes

photolyase, alkyltransferase and DNA-N-glycosylase

Photolyase

repairs thymine dimers

Alkyltransferase

repairs alkylated bases

Base excision repair (BER)

involves a category of enzymes known as DNA N-glycosylases

Nucleotide excision repair (NER)

can repair many types of DNA damage; is found in all prokaryotes and eukaryotes

Mismatch repair systems

found in both prokaryotes and eukaryotes and an important aspect of these
systems is that they are specific to the newly made strand; recognize and correct base pair mismatches

DNA double-strand breaks can be repaired by two systems that occur in both prokaryotes and eukaryotes

homologous recombination repair (HRR)
nonhomologous end joining (NHE)

Because sister chromatids are genetically identical

homologous recombination repair (HRR) can be an error-free repair mechanism

Non-homologous end joining (NHEJ) repair may result in

the deletion of a small region of the DNA.

When a replicative DNA polymerase encounters a damaged translesion DNA region

it is swapped for a TLS polymerase and the region is duplicated with error-prone replication

In the Holliday model for homologous recombination

break occurs at the identical location of two chromatids that are aligned together

In the double-strand break model

double-strand break creates a gap in the DNA of a single chromatid that must be repaired.

Gene conversion can occur in one of two ways

1. DNA mismatch repair
2. DNA gap repair synthesis

Gene conversion occurs in both

prokaryotes and eukaryotes

DNA mismatch repair can convert

one allele to another

Gap repair can convert

one allele to another

gene cloning

the technique of isolating and making many copies of a gene using a plasmid

Restriction enzymes

protect bacterial cells from invasion by foreign DNA, particularly that of a bacteriophage; bind to specific DNA sequences and then cleave the DNA at two defined locations, one on each strand

The sugar-phosphate backbone of DNA fragments with sticky or blunt ends generated by restriction enzymes

can be covalently linked together with DNA ligase

Reverse transcriptase

replicates RNA to DNA

The polymerase chain reaction (PCR)

is an in vitro method to amplify a specific sequence using directed DNA primers, DNA polymerase and dNTPS

One PCR cycle includes 3 steps

1.Denaturation
2.Primer annealing
3.Primer extension

After the third cycle of a PCR

the first complete product is formed

Reverse transcriptase PCR (RT-PCR)

is used to convert RNA to ds DNA

Real-time PCR or quantitative PCR (qPCR)

is used to quantitate the amount of mRNA that is expressed from a specific gene in a sample

dideoxy sequencing

involves the replication of DNA by a DNA primer, DNA polymerase and deoxyribonucleotides (dNTPs) in combination with dideoxyribonucleotides (ddNTPs) that terminate replication

second method of DNA sequencing

base-specific cleavage of DNA by chemicals

The resulting DNA sequence is read from

the smallest fragment to the largest fragment, since this corresponds to the order of the sequence from the primer

Just like in Sanger dideoxy sequencing the sequence in Maxam and Gilbert chemically induced base cleavage sequencing is read from

the bottom of the gel to the top of the gel

A single RNA in which the tracrRNA and crRNA are linked together
called single guide RNA (sgRNA)

can be created and used to do gene editing in any cell type.

spacer region

is designed that has homology to the targeted DNA to be edited

Southerns

The first blotting method to detect regions of DNA homology

Northern blotting

used to identify a specific mRNA and has many uses

Western blotting

used to identify a specific protein within a mixture of many protein molecules

The electrophoretic mobility shift assay (EMSA) or the gel retardation

used to determine if a protein binds to a specific DNA site or RNA molecule.

DNase I footprinting

used to determine the precise location of protein binding sites on DNA

A genetically modified organism (GMO)

an organism that has received genetic material via recombinant DNA technology

A non-GMO organism (non-GMO)

an organism that has not been modified using recombinant DNA technology

transgenic organism

An organism that has received genetic material from a different species

transgene

A gene from one species that is introduced into another species

Historically, insulin was isolated from cows and human cadavers

But now, patients can use insulin made by recombinant bacteria

in the A-chain

There is 1 intramolecular disulfide bond

between the A-chain and the B-chain

There are 2 intermolecular disulfide bonds

Biological control

use of microorganisms or their products to alleviate plant problems, such as disease or damage from environmental conditions

Agrobacterium radiobacter

can prevent crown gall disease caused by Agrobacterium tumefaciens

Bacillus thuringiensis toxins

lethal to many caterpillars and beetles but harmless to plants and humans

bioremediation

the use of microorganisms to reduce environmental pollutants

vaccine

a biological preparation that provides active acquired immunity against a disease

a vaccine consists of

an antigen derived from one or more components of the disease-causing agent that elicits an immune response, both antibodies to recognize and destroy the disease-causing agent and memory cells that enable the host to recognize and act against the disease-causing agent in the future

Innactivated vaccines

consist of an inactivated pathogen that cannot cause a disease

Attenuated vaccines

consist of an avirulent, but live pathogen

Viral vector vaccines

consist of a genetically modified avirulent virus that protects against a viral infection

Subunit vaccines

consist of purified components from a pathogen:
Protein vaccines
Polysaccharide vaccines
Nucleic acid vaccines
DNA plasmid vaccines
mRNA vaccines

Gene addition or gene knockin

adds a new gene into the genome

Gene deletion or gene knockout

deletes a gene from the genome

Cloning

the copying of a gene into a vector so multiple copies of the gene can be produced

Reproductive cloning

the production of two or more genetically identical individuals

Totipotent cells

like fertilized eggs, can give rise to all cell types

Pluripotent cells

like embryonic cells, can differentiate into almost every cell, but can’t give rise to an entire, intact individual

Multipotent cells

like hematopoietic cells in the bone marrow, can differentiate into
several cell types

Unipotent cells

like the germ cells in the testis, can only differentiate into one cell type

Adults contain only

multipotent or unipotent stem cells.

Multipotent hematopoietic stem cells (HSCs)

from bone marrow can be differentiated into several different cell types

Pluripotent embryonic stem cells (ES) and pluripotent embryonic germ cells (EG)

can easily be grown in the laboratory and provide the greatest potential for stem cell transplantation therapy

the Monsanto Company has produced corn plants that are highly
tolerant of glyphosate, the active ingredient in the Monsanto herbicide
Roundup.

Compared to nontransgenic plants, these transgenic plants grow
quite well in the presence of glyphosate-containing herbicides