Genetics

1
Genetics

• Genetics is the study of heredity
• What genes are
• How they are replicated
• How they carry information
• How they are passed to subsequent generations or
  passed between organisms

2
Genetic organization

• All the genetic information in a cell is the
  genome
• A cells genome is contained in its chromosomes
• Chromosomes contain genes
• Genes are segments of DNA (in viruses can be RNA)

3
Bacterial chromosomes

• Single circular strand of DNA
• There is only one per cell (Bacteria have a
  haploid (single) number of chromosomes
• Contained in the nucleoid region of the cell
  since there is no nuclear membrane
• Not associated with histone proteins (eukaryotic
  DNA does)
• Extrachromosomal pieces of DNA found in bacteria
  are known as plasmids

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Eukaryotic chromosomes

• Exist in pairs
• Have a diploid (2N) or haploid number of
  chromosomes (ex body cell 46, sperm cell 23)
• Exist as linear molecules of DNA
• Present in the nucleus which is enveloped in a
  nuclear membrane that contains pores
• Are associated with histone proteins
• Extrachromosomal DNA can be found in
  mitochondria/chloroplasts

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Comparison of DNA in prokaryotes and eukaryotes

• Both prokaryotes and eukaryote DNA is supercoiled
  and tightly packed
• DNA in eukaryotic cells is much longer (at least
  10X)
• DNA is a macromolecule composed of repeating
  units of nucleotides
• The inherited nature of DNA is found in the order
  of sequence of the nucleotides

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This unit will cover

• DNA
• Replication Transcription Mutation
• mRNA
• translation
  recombination
• polypeptides

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DNA

• Composed of monomer units called nucleotides
• Nucleotides are composed of three parts
• Phosphate group (PO4)
• Sugar- deoxyribose
• Nitrogenous base
• Adenine-Thymine
• Guanine-Cytosine
• Pairing rule dictates this arrangement
  -(complementary)
• (AT) (GC) 100

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DNA

• Exists in the shape of a twisted ladder known as
  a double helix (Watson and Crick, 1953)
• Is an antiparallel molecule- cut in half
  lengthwise, the DNA runs in opposite directions
• 5 ? 3 top to bottom
• Other is 3 ?5 from top to bottom

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DNA

• End with the hydroxyl attached to the 3 Carbon
  on the sugar is called the 3 end of the DNA
  strand
• End having a phosphate to the 5 carbon of the
  sugar is the 5 end

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DNA replication

• Replication is semi-conservative
• Each new strand of the double stranded DNA
  contains one original conserved strand and one
  new strand crating two identical daughter
  molecules
• Free nucleotides are present in the cytoplasm and
  pair with their complement on the original
  strands
• DNA polymerase synthesizes a strand of new DNA
  using one of the parental strands as a template

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RNA

• Made of nucleotide monomers also but differs from
  DNA in the following ways
• Ribose is the sugar
• Uracil replaces thymine and is a complementary
  base to adenine
• Single stranded molecule
• Much shorter strand than DNA

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RNA/DNA

• In eukaryotic cells, RNA exists inside the
  nucleus and also in the cytoplasm
• In eukaryotes, DNA exists in the nucleus and the
  mitochondria or chloroplasts
• In prokaryotes, RNA is mRNA, rRNA and tRNA

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Protein synthesis

• Is directed by DNA
• Transcription takes the directions from DNA to
  the cytoplasm
• Translation places the amino acids in the correct
  primary structure (correct order) to make each
  protein in the cell

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Transcription

• Is the synthesis of a complementary strand of RNA
  from a DNA template (mRNA)
• mRNA carries the coded information from DNA to
  the ribosomes where proteins are synthesized
• A G on the DNA is a _______ on the mRNA
• An A on the DNA is a _____ on the mRNA
• DNA strand A-G-T-C-G-A
• mRNA strand __-__-__-__-__-__

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Transcription

• Uses the enzyme RNA polymerase and RNA
  nucleotides from the cytoplasm for construction
  of the mRNA molecule

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Translation

• Protein synthesis occurs when the language of
  nucleic acids is decoded and converted to the
  language of proteins
• mRNA language is in the form of codons, groups of
  three nucleotides which determine the seq2uence
  of the amino acids
• There are 20 amino acids and 64 possible codons
  which means that there is more than one codon for
  each amino acid

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Translation

• Leucine has six
• UUA
• UUG
• CUU
• CUC
• CUA
• CUG

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Translation

• Sense codons code for amino acids
• Nonsense codons also called stop codons signal
  the end of a protein molecules synthesis
• Codons of the mRNA are read in sequence and the
  correct amino acid is assembled into the growing
  chain in response
• Site of the translation is the ribosome

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Translation

• tRNA molecules recognize the codon and transport
  the required amino acid to the ribosome
• tRNA has an anticodon which is a sequence of
  three bases that is complementary to the codon so
  that it can base pair with it and carry on its
  other end the amino acid that the tRNA recognizes

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Protein formation

• Amino acids are joined by peptide bonds by the
  ribosome
• Ribosome moves along the mRNA to the next codon
• Translation ends when one of the three nonsense
  codons in the mRNA molecule is reached
• PRACTICE!!!!

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Differences in prokaryotic and eukaryotic protein
synthesis

• In prokaryotes
• There are a number of ribosomes attached to a
  single mRNA, all at different stages
• Translation of mRNA can begin before
  transcription is complete because mRNA is being
  made in the cytoplasm

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Eukaryotes

• In eukaryotes
• Transcription occurs in the nucleus
• mRNA must move through the membrane into the
  cytoplasm
• mRNA undergoes processing before it leaves the
  nucleus
• Eukaryotic genes are made of exons, regions of
  DNA expressed and introns, intervening regions
  that do not encode proteins

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Eukaryotes

• RNA polymerase will synthesize the RNA with exons
  and introns
• Ribozymes will remove the intron derived RNA and
  splice together the exon derived RNA to produce
  mRNA

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Intermicrobial transfer of genetic information

• Generally beneficial to bacteria which can
  acquire new genes from other bacteria that adapts
  them for survival in a new environment
• Conjugation- genetic material is transferred from
  one bacterium to another
• Mediated by one kind of plasmid, circular DNA
  which replicates independently of the cells
  chromosome
• Requires direct cell to cell contact

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Conjugation

• Conjugating cells must generally be of the
  opposite mating type
• Donor cells must carry the plasmid, the recipient
  cell does not
• Gram plasmid codes for synthesis of sex pili
• Gram produce sticky surface molecules that cause
  cells to come into direct contact
• The F factor or fertility factor was the first
  plasmid transferred ( E coli) to be observed
  Donors (F) transfer plasmid to (F-) recipients

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Hfr

• In some cells carrying the F factor, the factor
  integrates into the chromosome converting the F
  cell to a Hfr cell (high freequency of
  recombination)
• Conjugation with Hfr cell, F- may acquire new
  versions of chromosomal genes but not the F
  factor. (just as in transformation)

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Plasmids

• F plasmids may contain additional genes
  transferred to the F- cell
• R plasmids have a significant medical importance
  R determinant has resistance genes which code
  for the production of enzymes to inactivate
  certain drugs or toxic substances (may be
  several)
• Plasmids which contain genes that increase
  pathogenicity of a bacteria Ex genes for a
  capsule
• Bacteriocinogenic- contain genes that synthesize
  toxic proteins that kill other bacteria
• Dissimilation plasmids which code for enzymes
  that trigger catabolism of unusual sugars and
  hydrocarbons Ex
  Pseudomonas uses toluene and camphor

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Transformation

• Genes are transferred from one bacteria to
  another as naked DNA
• Frederick Griffen, 1928 working on two strains of
  Streptococcus pneumonia
• One was pathogenic with a polysaccharide capsule
  to prevent phagocytosis
• Strain that lacked the capsule was not pathogenic
• Injected pathogenic heat killed bacteria in mice,
  there was no disease
• When he injected dead encapsulated bacteria mixed
  with non-encapsulated ones into mice, the mice
  died
• On necropsy, he found living encapsulated
  bacteria in the blood of the dead mice
• Avirulent bacteria were transformed into
  pathogenic ones

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Recombination

• A recipient cell with this new combination of
  genes is a kind of hybrid or a recombinant cell
• All descendants of this cell will be identical
• E coli is not naturally competent (does not
  naturally take up donor DNA) but lab treatment
  enables it to be used for genetic engineering

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Transduction

• Third mechanism of gene transfer
• Bacterial DNA is transferred from donor to
  recipient inside a virus called a bacteriophage
  (phage)
• In the lytic cycle of the virus, bacterial genes
  will be transferred to the newly infected
  recipient cells

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Mutations

• A mutation is a change in the base sequence of
  DNA or a change in the product encoded
• Can be beneficial when the mutant gene has a new
  or enhanced activity
• Can be harmful, even lethal
• Over 400 genetic human diseases are caused by
  mutations
• May involve one gene or entire chromosomes
• It is the driving force behind the theory of
  evolution

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Mutations

• Agents in the environment that directly or
  indirectly bring about mutations are called
  mutagens
• X-rays, U.V. radiation, chemicals, viruses and
  spontaneous replication errors are all examples
  of mutagens
• Many mutagens are also carcinogenic
• The Ames test uses bacteria as carcinogen
  indicators
• Mutant bacteria are exposed to mutagenic
  substances to cause a new mutation to reverse
  the effect (back mutations or reversions)

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Ames test

• Test measures the reversion of histadine
  auxotrophs
• An auxotroph has a nutritional requirement that
  is absent in the parent
• Salmonella typhinurium (his- to his)
• For mutagenic or carcinogenic activity to appear,
  the chemical to be tested, the his- cells and rat
  liver extract (rich source of activation enzymes)
  are incubated together
• If substance is mutagenic, the his- cells ? his
  at a higher rate than the spontaneous conversion

34
Mutation types

• Point mutations-
• the change in the DNA base sequence may cause no
  change in the activity of the product encoded by
  the gene (especially if it is in the third
  position of the mRNA codon
• Missense- when the mRNA is translated into
  protein, the incorrect base may cause the
  insertion of an incorrect amino acid Ex sickle
  cell anemia caused by a single base substitution

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Mutations

• Nonsense mutations- by creating a stop, or
  nonsense codon in the middle of an mRNA molecule,
  some base substitutions prevent the synthesis of
  a complete and functional protein
• Frameshift mutations-one or a few nucleotide
  pairs are deleted or inserted in the DNA
• Shifts the translational reading frame
• Almost always results in a long stretch of
  altered amino acids and the production of an
  inactive protein Ex Huntingtons disease is
  caused by extra bases inserted into a particular
  gene

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Genetic Engineering

• Genes are inserted into cells by a process coined
  genetic engineering
• Eukaryotic genes contain introns and exons (the
  transcript is converted to mRNA with the introns
  removed
• If such a gene is placed into a bacterial cell,
  it has no mechanism to remove the introns

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Step 1, 2

• Production of cDNA
• Create an artificial gene with only exons using
  reverse transcriptase to synthesize a
  complementary DNA from an mRNA template
• Use DNA polymerase to then synthesize a
  complementary strand of DNA
• Isolate R plasmids using density gradient
  centrifugation

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Step 3,4

• Join the cDNA and the R plasmids
• Plasmid o can be cut with the same restriction
  enzymes as the DNA to be cloned so that all the
  pieces of DNA will have the same sticky ends.
  When the pieces are mixed, the DNA to be cloned
  will become inserted into the plasmid
• Get the cDNA R plasmid into live bacterial cells
  by transformation (most cells are not competent
  so they are soaked in a solution of calcium
  chloride which makes them able to transform)

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Step 5

• Positive selection to ID the transformed bacteria
  
• Colony hybridization- DNA probes, or short
  segments of single stranded DNA that are
  complementary to the desired gene are
  synthesized. If the DNA probe finds a match, it
  will adhere to the target gene. The DNA probe has
  been labeled with a radioactive element or
  fluorescent dye so that its presence can be
  determined

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Step 6

• Clone the transformed bacteria
• Isolate and purify the human proteins that the
  bacteria produce for us
• Gamma interferon
• Insulin
• Factor VIII
• Erythropoietin
• Hep B vaccine
• Taxol

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Benefits attributed to genetic engineering

• Human genome project which mapped the
  35,000-70,000 genes in human DNA and sequenced
  the approximate 3 X 108 nucleotide pairs
  (completed in 2003)
• Understanding gene organization and control
• The Lac operon of gene expression consists of a
  set of operator and promotor sites and the
  structural genes they control

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Operon

• Combination of the three lac structural genes and
  the adjoining control regions is the lac operon
• Promotor is where RNA polymerase initiates
  transcription
• The operator acts like a stop or go signal for
  the transcription of the structural genes
• Based on the study of the induction of the
  enzymes of lactose catabolism in E coli