ch. 13 the molecular basis of inheritance

Rosalind Franklin (1952)

scientist who studied the DNA molecule

• developed an x-ray image of the DNA Helix

  • not credited for her work

  • enabled Watson and Crick to also study DNA molecule

James Watson and Francis Crick (1962)

scientists who were credited with the structure of DNA and researched the pairing of nitrongenous bases (who pairs with who)

how are nitrogenous bases paired?

purine (A & G) with purine (T & C)

Matthew Meselson and Franklin Stahl

scientists that studied DNA replication and that the two new strands are half old and half new

• semiconservative

origins of replication

where the two strands are separated

• opens up a replication bubbles

replication bubble in prokaryotes

• based off of ecoli

• only has one replication bubble

DNA replication in eukaryotes

• based on plants and humans

• has multiple replication bubbles

  • these eventually stretch out and combine

DNA polyermase and elongation

enzymes that add nucleotides only to the free 3’ end.

• meaning the new DNA strand can only elongate in the 5’-3’ direction

Helicase

separayes the two strands

topoisomerase

uncoild the DNA molecule (stretch out)

single-strand binding proteins

strengthens the templates

• keeps them stable

primase

adds the RNA primer

RNA primer

shows where DNA polymerase will start

• made of RNA nucleotides (no thymine)

  • gets replaced later (short lived)

DNA Polymerase 3 and 1

polymerase 3

• the enzyme that adds nucleotides and synthesizes the new strand

polymerase 1

• enzyme that replaces RNA primers with DNA nucleotides

leading strand

the template that is 3’ to 5’

• DNA polymerase will synthesize CONTINUOUSLY replication fork

  • 5’ to 3’

lagging strand

the template that is 5’ to 3’

• DNA polymerase must follow a 5’ to 3’ direction, so the new strand for this template is synthesized in chunks away from the replication fork

  • chunks are known as okazaki fragments

    • RNA primers must be placed each time

DNA ligase

the enzyme that glues the okazaki fragments together

replication bubble - lagging and leading strand

from the origin of replication the lagging strand and leading strand alternate

• this is because it depends which template it is on and which direction it is going

which enzyme proofreads and repairs DNA?

DNA polymerase

• the enzyme corrects eerors in base pairing

  • e.g. a thymine dimer (connection between nucleotides in the wrong way

    • polymerase checks for the error

      • a nuclease enzyme then cuts out the damaged DNA section

      • DNA polymerase repairs the cut out section with the missing nucleotides

      • DNA ligase will then glue the new section with the nucleotides around it

xeroderma pigmentosum (XP)

a disorder caused by an inherited defect in the nucleotide excision repair

• disorder occurs when damage caused by ultraviolet light that does not get corrected

  • individuals with XP are hypersensitive to sunlight

  • children who have XP can develop skin cancer (melanoma or basal carcenoga) by age 10 (moonlight kids)

  • mostly affects the eyes and areas of skin exposed to the sun

  • follow autosomal pattern of inheritance and effects about 1 in 1 million people in the U.S., but can be much higher in certain parts of the world

what does a chromosome consist of?

a DNA molecule that is packed together with proteins

bacterial chromosome

a double-stranded, circular DNA molecule associated with a small amount of protein

• in bacterium, the DNA is super coiled and found in a region called the nucleoid

eukaryotic chromosomes

have linear DNA molecule associated with a large amount of protein

• these proteins are called histones

histones

proteins that are responsible for the first level of DNA packing in chromatin

• it super coils the DNA

nucleosome

consists of DNA wound twice around a protein core of 8 histones

• these pack together into a chromosome (chromatids)

Frederick Griffith (1928)

scientist researched two types of Streptococcus pneumoniae (bacterium)

• Pathogenic (S - smooth)

• Harmless (R - rough)

  • concluded the Transformation factor

pathogenic (S cells)

have capsules that protect them from the animals’ immune system

• surrounded by protective coating known as smooth cells

  • causes the virus

harmless (R cells)

bacteria that do not have capsules

• no protective coating

  • does not cause the virus because the immune system will kill it

    • harmless and known as ROUGH cells

Griffith's experiment

gave mice different bacteria cells

1. mouse given S cells (control) → died

2. mouse given R cells (control) → lived (healthy)

3. mouse given heat-killed (killed by high temps; cannot work) S cells (control) → lived (healthy)

4. mouse given a mixture of heat killed S cells and living R cells → died (found living S cells in the mouse)

• concluded R bacteria, that normally be killed, transformed into S bacteria via an unknown, heritable substance (factor)

transformation

change in genotype and phenotype due to assimilation of foreign DNA by a cell

• S genotype got assimilated into R genotype (tranformed)

  • later concluded that DNA was the heritable factor that would cause tranformation

what does this show about viral DNA

• additional evidence that DNA was the genetic material came from studies of virus that infect other bacteria

  • such as bacteriophages (or phages)

phage

consists of a phage head with DNA inside, a tail sheath and tail fiber

• everything except the DNA is made of proteins

how do phages reproduce?

a phage will attach to a host cell's plasma membrane (with the fibers) and inject the DNA, leaving the protein behind

• the DNA will enter the host cell, while the original DNA gets destroyed

  • the enzymes and nucleotides in the host cell begin to replicate the phage DNA

    • other enzymes and ribosomes will manufacture phage proteins then assemble to form phages

    • a phage enzyme will then digest the bacterial cell wall and cause the cell to rupture (lysis)

• this lets the formed phages out, can be up to 200 phages, that will go to infect other cells

Alfred hershey and Martha Chase (1952)

researched T2 phages and whether or the protein carries the genetic info or the DNA does

• done with an E. coli host + T2 phages

  • concluded that DNA is the genetic material that is responsible for hereditary

phage experiment process

phages were laced with two different radioactive solutions

• one will be on the protein (pink)

• one will be on the DNA (blue)

  • this is to check which provides the genetic info

    • the pink one showed that the proteins stayed out of the host cell

      • the sample from the host cell showed no pink pigment

    • the blue one showed that the DNA went into the cell

      • the sample from the host cell showed blue pigment

what did the experiment conclude?

• DNA is the genetic material that causes hereditary

• injected DNA provides genetic info that makes the cell produce new T2 DNA and proteins

Erwin Chargaff (1950)

scientist that studied the DNA composition

• varies from one species to the next

  • developed Chargaff's rule

Chargaff's rule

in any species there is an equal number of A and T bases, and an equal number of G and C bases

Maurice Wilkins

scientist who worked w/ and took the DNA picture

• received nobel prize with Watson and Crick

Linus Pauling

proposed a three-stranded model of DNA

• this was an incorrect model of DNA

DNA cloning

yields multiple copies of a DNA segment

• uses bacteria and their plasmids

plasmids

small circular DNA molecules that replicate separately from the bacterial chromosome

• found in bacterial cells

what are plasmids used for?

to create more of the same gene or more of the protein produced by the gene

how do plasmids clone a gene of interest?

• plasmid from a bacterium has a gene of interest inserted into it

  • creates a recombinant plasmid (DNA)

    • plasmid will then be placed back into the bacterium

      • this will allow the gene of interest to be cloned

restriction enzymes

used to make recombinant DNA

• they cut DNA molecules at a specific DNA sequence called restriction sites

  • cuts the DNA that will have “sticky ends” that will bond with complementary “sticky ends" of other fragments (glue w/ glue)

    • DNA ligase seals the bonds betwen restriction fragments (super glue)

gel electrophoresis

used to sort DNA molecules/fragments that were produced by restriction enzymes

• restriction fragment analysis

  • can also be used to get for diseases such as sickle cell

  • also used for comparing forensic evidence

how does the gel electrophoresis work?

consists of a gel and a power source (a cathode end and an anode end)

• DNA fragments will be placed into the wells on the cathode side of the gel

  • these will run depending on their size

    • fragments closer to the anode end is very light and fragments closer to the cathode end is heavy

      • fragments are like unique fingerprints

• to observe the fragments the gel must be put in UV light

sickle cell DNA - restriction fragment analysis

when observed in gel electrophoresis:

• one of the restriction sites (fragments) is destroyed that comes from the DDeI enzyme

  • as a result the enzyme will generate different fragments when sickle cell DNA is present (will differ from normal DNA)

    • the fragment in sickle cell DNA combines 2 of the 3 fragments in normal DNA into one fragment

      • normal DNA has 3 fragments

      • sickle cell DNA has 2 fragments

        • this can be observed in a gel electrophoresis