© 2003 mark s. davis chapter 21 nucleic acids. © 2003 mark s. davis thanks mom… ishihara’s...

© 2003 Mark S. Davis

Chapter 21

Nucleic Acids


Thanks Mom…

• Ishihara’s simple test

• X-linked

• Easy to trace


25


What do you see?


Thanks Dad ??? (not really)• Thalassemia• Hereditary in Mediterranean people• Defective hemoglobin• Beta thalassemia results in an excess of alpha

globins, which leads to the formation of alpha globin tetramers (4) that accumulate in the erythroblast (immature red blood cell). These aggregates are very insoluble and precipitation interferes with erythropoiesis, cell maturation and cell membrane function, leading to ineffective erythropoiesis and anemia.


Queen Victoria

• Hemophilia

• Passed it on to several royal families in Europe


Goals

• Describe nucleotides, RNA and DNA, polypeptides

• Know the 3D structure of nucleic acids

• Describe mutations and their effects

• Describe viruses and Recombinant DNA technology


Chromosomes• Humans have 46

– Each cell is diploid

• Germ cells have 23– Called haploid– Contain one copy of each chromosome

• Contain all genetic information of the organism

• DNA


RNA

• Leave nucleus

• Direct protein synthesis


Nucleotides

• Monomers that make up DNA and RNA

• DNA – 2-deoxy-D-ribose

• RNA – D-ribose


Nucleotides

• Made from–Phosphoric acid

–Sugar

–Purine – bicyclic

–Pyrimidines – single ring


Naming• Sugar and base

–Adenine + sugar = Adenosine

–Cytosine + sugar = Cytidine

• 5’ – monophosphate is second part of the name

• dGMP


5’ – 3’ Phosphodiester

• In making DNA – dehydration occurs at C3 and phosphate group on C5

• This is the direction used for naming


DNA Structure

• Watson and Crick 1953

• Double helix – right handed

• Complementary pairs–A-T

–G-C


Complementary Strands

• Bases located opposite

• Maximum hydrogen bonding

• Maximum stability

• Hydrophobic bonds to the bases above and below


Given…

• G A T T A C A

• What is the complementary strand?


Size of DNA

• 3x109 base pairs• Stretch to 1m in length• Extremely compact • Organized around histones• Around 200 base pairs/histone


RNA

• Single strand

• Different types–tRNA

–mRNA

–rRNA


RNA Pairing• A-U

• G-C

• Only about ½ molecules base pair

• Acceptor stem: 3’ end

• Anticodon - opposite


Information flow

• Replication: copy DNA in cell division

• Transcription: make RNA (all 3 types) from DNA

• Translation: make polypeptides using rRNA, mRNA, tRNA


Replication

• Copy all 46 chromosomes in less than a day (about 8 hrs)

• Error – less than 1 in 10 billion• Always in 5’ 3’ direction and two

strands grow opposite• DNA Polymerase catalyzes


DNA Polymerase

• Catalyzes pairing of new bases

• “Checks” the accuracy of the pairing and correcting errors

• Parent DNA gives rise to two “daughter” duplexes


Practice

• What is the corresponding daughter strand to the parent – 5’-TAGTTCGT-3’


Transcription

• Transcription bubble

• RNA Polymerase acts on the template strand only

• There is a start site

• There is a termination site


RNA Polymerase

• No proofreading function

• Error less than 1 in 100,000

• Initial RNA is called primary transcript RNA, ptRNA

• Later modified to the other types


What RNA is formed?

• If the DNA sequence is: 5’-GCCTAG-3’

• What is the RNA that is synthesized?


Post transcription

• End capping– Adds particular units to the 5’ and 3’ end

• Base modification

• Splicing– Deletes introns– Keeps exons


Translation• Protein synthesis

– mRNA, tRNA and rRNA involved– Occurs in cytosol at Ribosomes

• Sequence of bases specifies amino acid sequence– Three bases (codon) = one amino acid

• 64 codons for 20 amino acids– Met, only one codon (START codon)– Stop by three codons


Translation• Each tRNA carries ONLY ONE aa

• Aminoacyl-tRNA synthetase– Bonds aa to tRNA at 3’ end (ester linkage)

• Peptidyl transferase– Catalyzes transfer of amino acid from tRNA

at ribosome

• Synthesis terminates when STOP codon is reached


Posttranslational Processing

• Most lose Met (start residue)

• Folding begins

• Disulfide-bridging

• Quaternary structures assembled


Control at every step

• Not every cell expresses every gene

• Specialized

• Repressor proteins– Turn off synthesis of “unneeded” proteins

• Inducer proteins– Turn on synthesis of needed proteins


Mutations• Error in base sequence

– Alteration of protein function– Loss of protein function– No noticeable change

• Substitution (point) mutations– One base in place of another

• Frameshift mutations– Base added or deleted


Spontaneous Mutations and Mutagens

• Spontaneous mutations– Errors in replication

• Sodium nitrite– In processed meats– Converts cytosine to uracil– Overall danger thought to be low– Reduces occurrence of botulism


Spontaneous Mutations and Mutagens

• Benzopyrene– Causes frameshift mutation– Found in car exhaust, tobacco smoke,

burnt meats

• Radiation– Can break bonds and modify bases and

base sequences


Silent Mutations

• Base-sequence errors that don’t affect organism– 64 codons for 20 amino acids– Similar amino acids can be substituted– Change may be in unimportant region– Mutation located in intron– Genes have 2 or more copies


Mutations

• Somatic cells– Affect individual organism

• Germ cells– Affect all offspring– Genetic (hereditary) diseases


Antibiotics

• Chemicals to fight infection

• Block protein synthesis – Stop replication– Attack cell walls

• Must finish whole course

• Bacteria mutate quickly

• Antibiotic resistant bacteria


Viruses

• DNA or RNA with protein coat

• No functions outside cell

• Enter cell and “hijack” it

• Each virus attacks only specific cells– TMV– AIDS


DNA Viruses

• Enter host cell and nucleus

• Insert themselves into host genome

• Hiding inside cell – hard for immune system to detect

• Can stay indefinitely


RNA Viruses

• Enters cell

• Directs synthesis of RNA replicase in cytosol

• Uses machinery of host to make copies of itself


Retroviruses

• Special RNA virus

• Enters cell and directs synthesis of viral DNA using reverse transcriptase

• DNA inserts into host genome

• Can hide or remain dormant for long periods of time– HIV


Treatment

• Antibiotics don’t work

• Body doesn’t recognize virus once hiding in host cell

• Best method: preemptive vaccines


Recombinant DNA Technology

• Began in mid-70s

• Transplanting or altering of DNA

• Benefits– Therapeutic drugs– Improvements to crops and herds– Curing/treating of genetic diseases


Production of human insulin

• First application of recombinant DNA technology

• Uses yeast and bacteria as vehicle– Bacteria have genomic DNA and a

plasmid

• Less side effects than cow or pig insulin


ProcessIdentify gene encoding wanted protein

1. Isolate this gene from the donor DNA

2. Splice into plasmid (vector DNA)1. Restriction enzymes

3. Recombinant DNA (new plasmid) back into E. coli

1. Chemical shock

2. Heat shock


Other techniques• Microinjections

– Direct injection of DNA into nucleus of another cell

– Cloning ~~~ Dolly

• Viral vectors– Altered virus (usually retrovirus)– Carries new DNA to host cell– Research now for cystic fibrosis


A Little More About Cloning• Enucleation of cell:

– http://gslc.genetics.utah.edu/units/cloning/whatiscloning/images/enucleation.mpg

• Nuclear Transfer:– http://gslc.genetics.utah.edu/units/cloning/

whatiscloning/images/transfer.mpg

• Cloning “Practice”:– http://gslc.genetics.utah.edu/units/cloning/c

lickandclone/


Transgenic Breeding

• Organisms with altered DNA

• Grow faster, larger, etc.

• Resistant to pests– http://www.pbs.org/wgbh/harvest/engineer/t

ransgen.html

• Many already in your supermarket


Gene Therapy

• Human Genome Project– Finished with sequence– Now identify genes and proteins

• Insert correct gene for defective one• Modified adenovirus (common cold)

– Aerosol spray inhaled– Injection into bloodstream– Incubation of cells


Ethical Considerations• Effects of recombinant DNA?

• Can we test people for diseases?– Alzheimer’s; Huntington’s

• Gene Therapy– Enhance intelligence, strength– Pick eye color

• Who will benefit? Will anyone suffer?


Internet Sites of Interest

• PBS Site about GMOs– http://www.pbs.org/wgbh/harvest/

• Genetic Science Learning Center– http://www.pbs.org/wgbh/harvest/

• NWABR– http://www.nwabr.org

© 2003 mark s. davis chapter 21 nucleic acids. © 2003 mark s. davis thanks mom… ishihara’s...

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