Protein structure

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• Protein structure
• Polypeptide and proteins- Both described
  molecules composed of amino acids. But they
  differ in their state of assembly and functional
  capacity. As assembled on the ribosome during
  translation the molecule is named as polypeptide.
  After released from the ribosome the polypeptide
  folds up and assumes a higher order structure.
• The two most common secondary structure
  arrangements are the right-handed a-helix and the
  b-sheet, which can be connected into a larger
  tertiary structure (or fold) which defines the 3
  dimensional conformation of the entire chain in
  space.
• When the final conformation is achieved only the
  molecule becomes functional and is called a
  protein. It is the 3 dimensional conformation
  that is essential to the function of the protein

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• The quarternary level of organization applies
  only to proteins composed of more than one
  polypeptide chains. This type of protein is
  called oligomeric and each chain is called a
  subunit eg. Hemoglobin, DNA and RNA polymerase
  have quarternary structures.
• Posttranslational modifications of proteins
• The N-terminus and C terminus amino acids are
  usually removed or modified. Eg. N terminal
  formylmethionine of bacterial polypeptide is
  usually removed.
• Individual amino acid residues are sometimes
  modified. Addition of methyl groups or phosphates
  to the hydroxyl group Kinases are responsible for
  addition of PO4.
• Carbohydrate side chains are sometimes added,
  glycoproteins
• Polypeptide chains are often complexed with metal
  ions.

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• Electrophoresis of nucleic acids
• Electrophoresis is a technique used to separate
  macromolecules - especially proteins and nucleic
  acids - that differ in size, charge or
  conformation. As such, it is one of the most
  widely-used techniques in biochemistry and
  molecular biology.
• When charged molecules are placed in an electric
  field, they migrate toward either the positive
  (anode) or negative (cathode) pole according to
  their charge. In contrast to proteins, which can
  have either a net positive or net negative
  charge, nucleic acids have a consistent negative
  charge imparted by their phosphate backbone, and
  migrate toward the anode.
• Proteins and nucleic acids are electrophoresed
  within a matrix or "gel, agarose or
  polyacrylamide
• Ethidium bromide, a fluorescent dye is used for
  staining nucleic acids and DNA bands are visible
  under UV light.

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• Recombinant DNA Technology
• DEFINITION OF RECOMBINANT DNA
• DNA molecules constructed outside of living cells
  by joining natural or synthetic DNA segments to
  DNA molecules that can replicate in a living cell
• Restriction endonuclease Any one of several
  enzymes, produced by bacteria, that break foreign
  DNA molecules at very specific sites. Extensively
  used in recombinant DNA technology. Restriction
  Enzymes are made to protect the bacteria from
  foreign DNA. Bacteria have a method of marking
  their own DNA as being "self" (called a
  Modification System). Any DNA not recognized as
  self is digested into smaller pieces by the
  Restriction Enzymes
• Recognition site The specific site at which
  restriction endonucleases cleave DNA is defined
  by a sequence of bases.

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• Restriction Enzymes search for exact sequences of
  a defined length (recognition sites). Some
  enzymes recognize sequences 4 bp long (e.g.,
  GTAC), some 6 (e.g., GAATTC), and still others 8
  or more.

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• One of the common features of most enzyme
  recognition sites is that they are palindromes. A
  palindrome is a sequence which is read the same
  on both strands in the 5' --gt 3' direction.
• For Res. Enzyme EcoRI 5 GAATTC 3
• 3
  CTTAAG 5
• 
  
• This type of cut is called staggered, because it
  results in fragments with single-stranded ends.
  The single-stranded ends are said to be sticky
  because they are able to bind to a complementary
  single-stranded region
• According to the pattern of cleavage Res. Enzymes
  results in 3 types of DNA fragments
• 5 overhangs 5 G.----------3
• 3 CTTAAG 5

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• 3 overhangs 5 GAATTC 3
• 3 C---------- 5 these
  make sticky ends
• Blunt ends 5 GTT.3
• 3 CAA..5
• Different Res. Enzymes recognize and cleave
  different of bases
• Eg. 4 base cutters, 5,6,7 etc ex. EcoRI is a 6
  base cutter
• How often will a RE cut a random DNA fragment?
• A 4 base cutter 1/4x1/4x1/4x1/4 1/256 bp of DNA
  (one in every 256 base pairs of DNA)
• The general rule is (1/4)n where n is the of
  nucleotide bases in the restriction site.
• These sticky ends can reanneal with complementary
  single stranded tails on other DNA fragmets. If
  mixed under the proper conditions, DNA fragments
  from two sources form recombinant molecules and
  DNA ligase links the two fragments.

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• DNA cloning and cloning vectors
• What is molecular cloning? Genetically
  engineered replicates of DNA sequences
• Why clone DNA ?
• To identify an individual gene
• To isolate a gene
• To clone genomic DNA
• To make clones made from mRNA, called cDNA clones
• To construct genomic libraries/cDNA libraries
• Genomic libraries are collections of clones that
  may contain at least one copy of every DNA
  sequence from the genome of interest.
• Cloning vectors Vehicles that enable the
  replication of cloned fragments. Most have some
  general features in common.
• - small and well characterized genomes that
  contain an origin of replication to enable the
  molecule to replicate itself and the insert
  fragment.

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• All cloning vectors must have 4 common features
  
• An origin of repliation (Ori site)
• A dominant selectable marker, usually for
  antbiotic resistance I.e.
• Unique restriction enzyme sites should be
  available
• Must be easy to recover from a bacterial host
• Plasmids- These are molecules of DNA that are
  found in bacteria separate from the bacterial
  chromosome. They
• are small (a few thousand base pairs)
• usually carry only one or a few genes
• are circular ds DNA molecules,
• have a single origin of replication
• In nature, these plasmids often encode genes for
  proteins (e.g., enzymes) that protect the
  bacterium from one or more antibiotics.

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• Plasmids were The first vectors to be genetically
  modified and used in cloning
• To use them as vectors they should be modified
  to contian a limited number of res. Sites and
  selectable marker genes that detect the presence
  of plasmid in the host cell.
• Selectable markers
• When you transform bacteria, not all cells will
  receive copies of the plasmid
• Selectable marker genes allow for easy
  identification of transformed cells
• The most common selectable markers are antibiotic
  resistance genes. ampicillin (amp), tetracyclin
  (tet) or Cloramphenicol (Cam) or Kanamycin (Kan)

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• Cloning sites
• These are regions of DNA which contain the
  consensus sequence for numerous restriction
  enzymes.
• Called the multiple cloning site (MCS) or
  polylinker
• The restriction enzymes that cut within the MCS
  do not cut elsewhere in the vector
• This allows for target DNA to be inserted into a
  specific part of the vector.
• Screening of recombinants- once the DNA of
  interest is inserted to the plasmid at the
  restriction site in the polylinker the
  recombinant plasmid then have to be transformed
  into bacteria. Within the bacteria these plasmids
  produce large number of copies through its
  replication process.
• Bacteria cells are plated on media containig the
  appropriate antibiotic, IPTG (a lactose analog)
  and X-gal. When induced by IPTG the galactoside
  gene will be transcribed and the enzyme will

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• Use X-gal as a substrate.
• When X-gal is cleaved it produces a blue pigment.
  When there is an insert in the polylinker lacZ
  transcription is disturbed no B galatosidase is
  formed and the X-gal not cleaved.
• Colonies with inserts will be white

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• One such plasmid is pUC18, which is small (2686
  bp) and allows to carry relatively large DNA
  inserts. In a host cell it replicates 500 copies
  per cell, producing many copies of inserted DNA
  fragments.
• A large of restriction sites are available in a
  polylinker site.
• It has a selection system to identify the
  recombinant plasmids.
• The pUC 18 carries the bacterial lacZ gene. When
  grown in a medium of X-gal it produces a blue
  colony. The polylinker is inserted within the
  lacZ gene. When a DNA fragment is inserted into
  the polylinker site, the lacZ gene is
  inactivated. Therefore the recombinant plasmids
  when grown in X-gal would produce white colonies.
  
• One disadvantage in plasmid vectors is it can
  carry up to 10 kb of inserted DNA. For
  experiments when larger DNA fragments are
  required genetically modified strains of lambda
  phage are used as vectors.

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• Cloning in E.coli host cells- one of the most
  commonly used hosts is a laboratry strain of
  E.coli known as K12.
• Lambda phage vectors are ds virusDNA that
  infect E.coli cells. These bacteriophage ?
  cloning vectors has a chromosome with a left and
  right arm that collectively contain all the
  essential genes for the lytic cycle. Between the
  two arms is a dispensable segment of DNA with
  EcoRI sites. This dispensable region is replaced
  by the DNA insert. They are then inserted into
  bacterial host cells where they reproduce to form
  many infected particles of phages, each carrying
  a DNA insert. Phage vectors can carry about 20 kb
  DNA inserts, and useful when cloning entire
  genomes. Fig 16.9

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• . Cosmids Cosmids are vectors which have
  characteristics of both viruses and plasmids.
  They contain the phage cos site necessary for
  packaging DNA into phage heads. Most of the
  lambda genes have been deleted.
• 1) Can accept 40-50 kb inserts.
• 2) Recombinant cosmids are packaged into phage
  heads and the particles used to infect cells.
• 3) Once inside a cell, the DNA replicates as a
  plasmid
• . Shuttle Vectors
• 1) Designed to replicate and function in two
  different kinds of cells
• 2) E. coli and yeast
• 3) E. coli and Agrobacterium
• Cloning in Eukaryotic host cells The yeast
  Saccharomyces cerevisiae is extensively used as a
  host cell for growth and expression of cloned
  eukaryotic genes.

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• Yeast Artificial chromosomes (YAC)
• Eukaryotic chromosomes have common features that
  enable them to successfully replicate in a cell.
  They have
• Two telomeres
• A centromere
• An origin of replication along the chromosomes
  (ARS)
• YAC are cloning vectors that have been
  constructed so that you can add inserts between
  the telomeric regions and effectively clone very
  large regions of DNA.
• YAC vectors have A selectable marker at each
  end TRPI (tryptophan independence) and URA3
  (Uracil independence) and a restriction enzyme
  site.
• Using the above procedures, many human genes have
  been cloned in E. coli or in yeast. This has made
  it possible - for the first time - to produce
  unlimited amounts of human proteins in vitro.
  Cultured cells (E. coli, yeast, mammalian cells)
  transformed with the human gene are being used to
  manufacture

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• Insulin for diabetics
• Factor VIII for males suffering from hemophilia A
  
• Factor IX for hemophilia B
• Human growth hormone (GH)
• erythropoietin (EPO) for treating anemia
• three types of interferons
• several interleukins
• adenosine deaminase (ADA) for treating some forms
  of severe combined immunodeficiency (SCID)
• Parathyroid hormone and many more
• Cloning without host cells
• Polymerase Chain Reaction (PCR) Developed in
  1986 this is a rapid method of DNA cloning and
  very much used instead of vectors

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• Polymerase chain reaction is a technique that
  amplifies DNA, which enables to make millions -
  or even billions - of copies of a DNA molecule in
  a very short time.
• PCR has been the most widely used technique in
  the field of molecular biology since it was
  developed in 1986.
• It has been used to detect DNA sequences, to
  diagnose genetic diseases, to carry out DNA
  fingerprinting, to detect bacteria or viruses
  (particularly the AIDS virus), and to research on
  human evolution. It has even been used to clone
  the DNA of an Egyptian mummy!
• PCR Methodology
• In order to perform PCR, you must know at least a
  portion of the sequence of the DNA molecule that
  you wish to replicate
• You must then synthesize primers short
  oligonucleotides (containing about two dozen
  nucleotides) that are precisely complementary to
  the sequence at the 3' end of each strand of the
  target DNA (DNA you wish to amplify ) (Fig.
  16-11)

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• The DNA sample is heated to separate its strands
  denaturation (denature at 90-95c) and mixed with
  the primers-.
• Temp. is then lowered to 50-70c when primers
  would anneal, if they find their complementary
  sequences in the flanking regions of the target
  DNA- annealing.
• Synthesis begins (as always 5' -gt 3') using the
  original strand as the template(Temp. between
  70-75c).
• The reaction mixture must contain all four
  deoxynucleotide triphosphates (dATP, dCTP, dGTP,
  dTTP), DNA polymerase and Mg2. A special kind
  of DNA polymerase is used that is not denatured
  by the high temperature needed to separate the
  DNA strands, the enzyme Taq polymerase is
  obtained from the heat stable bacteria Thermus
  aquaticus
• Taq polymerase extends the primers and
  polymerization continues until each
  newly-synthesized strand has proceeded far enough
  to contain the site recognized by the other
  primer- extension.

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• In this way two DNA molecules identical to the
  original molecule is formed and the cycle is
  repeated.
• Each cycle doubles the number of DNA molecules.
• Using automated equipment (Thermocycler), each
  cycle of replication can be completed in less
  than 5 minutes. After 30 cycles, what began as a
  single molecule of DNA has been amplified into
  more than a billion copies (230 1.02 x 109).
• Advantages of PCR
• Requires very minute amounts of DNA
• Little DNA purification is needed.
• Partially degraded DNA can be used
• Extremely rapid diagnostic potential and very
  sensitive
• Amenable to many analytical procedures
• Can be largely automated

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• Limitations in PCR
• Generated sequences may have errors
• False results possible if DNA is contaminated
• Some applications have reproducibility problems

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