Unit 4

Unit 4

CHAPTER 16: CONTROL OF GENE EXPRESSION
1. CONTROL OF GENE EXPRESSION
  1. PROKARYOTES
    1. regulate gene expression in response to their environment
    2. OPERONS
      1. REPRESSION (makes product)
        if product is available, enzymes are not needed
        trp OPERON
        genes associated with the amino acid TRYPTOPHAN
        coding region has 5 ENZYMES
        REPRESSOR binds to OPERATOR when
        TRYPTOPHAN is present
        tryptophan binds to repressor and changes its shape
        so it CAN BIND to operator
      2. INDUCTION (makes enzyme)
        made in response to substrate
        lac OPERON
        REPRESSOR binds to OPERATOR when
        ALLOLACTOSE is NOT present
        allolactose binds to repressor and changes its shape
        so it CANNOT BIND to operator
        GLUCOSE REPRESSION
        bacterium prefer glucose, and want to break it down first
        (easier to do)
        they break down lactose when there is no more glucose
        high levels of glucose = low levels of cAMP
        low levels of glucose = high levels of cAMP
        high levels of cAMP enables cAMP to bind to CAP to form complex
        enzymes are NOT needed if glucose is present
      3. 2 regions:
        regulatory region
        controls whether coding region is transcribed or not
        PROMOTER
        GENE
        CAP-BINDING SITE
        PROMOTER
        OPERATOR
        REPRESSOR
        repressor may bind to operator to STOP
        CONFORMATIONAL CHANGE
        shape of repressor changes
        RNA polymerase attaches to promoter
        binding site for catabolite activator protein
        helps RNA polymerase bind to promoter
        cAMP binds to site to form complex
        gene for repressor protein
        promoter for repressor gene
        coding region
        code for enzymes
        GENES
  2. EUKARYOTES
    1. regulate gene expression to maintain homeostasis
    2. TRANSCRIPTION FACTORS
      1. important for RNA to bind
      2. GENERAL
        helps bind RNA polymerase
        promoters
      3. SPECIFIC
        increases level of transcription; makes process better; enhances
        activator binds to enhancer, forms loop
2. GENE REGULATION
  1. REGULATORY PROTEINS regulate gene expression
    1. proteins bind to specific sequences of DNA
    2. regulatory proteins possess DNA-binding MOTIFS
      1. DNA-BINDING MOTIFS
        regions of regulatory proteins which bind to DNA
3. POSTTRANSCRIPTIONAL REGULATION
  1. gene expression regulated after transcription
    1. ALTERNATIVE SPLICING
      1. introns spliced out of pre-mRNA
    2. RNA EDITING
      1. chemical modifications
      2. NOT A MUTATION: not in genome, in RNA
CHAPTER 19: CELLULAR MECHANISMS OF DEVELOPMENT
1. PROCESS OF DEVELOPMENT
  1. CELL DIFFERENTATION
    1. CELL DIVISION
      1. EMBRYOGENESIS
        Development of embryo; before birth
        series of mitotic division after fertilization to increase amount of cells
    2. PATTERN FORMATION
      1. MORPHOGENESIS
        MORPH : SHAPE
        GENESIS : PRODUCE
        ANIMALS REGULATE
        number, timing, and orientation of cell division
        growth and expansion
      2. first more cells need to be made
        cells adopt fate based on location
    3. all cells within an individual organism are the same; they all have the same DNA (genetic information)
      1. CELL DETERMINATION
        molecular decision to become a particular type of cell
        cells already know what type of cell they will become
        acquires positional label that reflects its location in the embryo; whats around cell may influence cell
        CELLS BECOME COMMITTED
        DETERMINATION takes place in STAGES
2. STEM CELLS
  1. PLURIPOTENT
    1. TOTIPOTENT
    2. MULTIPOTENT
      1. UNIPOTENT
CHAPTER 17: BIOTECHNOLOGY
1. RESTRICTION ENDONUCLEASES
  1. RECOMBINANT DNA
    1. single DNA molecule made from two different sources
  2. GEL ELECTROPHORESIS
    1. separates DNA fragments by size
    2. MOLECULAR CLONING W/ VECTORS
      1. VECTOR
        carries something from one place to another
      2. REVERSE TRANSCRIPTASE
        DNA LIBRARIES
        collection of DNA molecules that can be MAINTAINED and REPLICATED in a HOST ORGANISM
        DNA inserted into PLASMID then inserted into BACTERIA
        CLONING VECTORS NEED:
        a sequence that allows replication in host organism
        a selectable marker
        sequences that allow DNA fragments to be added
        cDNA LIBRARIES
        POLYMERASE CHAIN REACTION (PCR)
        mimics DNA REPLICATION to produces MILLIONS of COPIES of a DNA sequence
        allows amplification via PRIMERS
        3 STEPS in PCR
        DENATURATION
        ANNEALING OF PRIMERS
        DNA SYNTHESIS
        DNA polymerase attaches to primer, synthesizes DNA
        72
        RNA PRIMERS BIND to DNA fragment
        55
        PRIMERS ADDED
        HEAT SEPARATES 2 strand DNA into SINGLE strand DNA
        95
        put into THERMAL CYCLER
        REVERSE TRANSCRIPTION PCR (RT-PCR)
        PCR is performed on cDNA made from RNA
        REVERSE TRANSCRIPTION to make cDNA
        cDNA is then used in PCR
        QUANTITATIVE (RT-PCR)
        involves isolating mRNA, converting to cDNA using RT, then using PCR to amplify specific cDNAs
        then amount of DNA produced is measured in real time
        dyes added to DNA to be visualized
        DNA FINGERPRINTING
        can identify individual with small amount of tissue or body fluids
        viruses use REVERSE TRANSCRIPTASE to use RNA to make DNA
        BACTERIA DO NOT CUT INTRONS OUT
      3. bacteria have 1 circlular chromosome
        PLASMID
        not essential for survival; bonus material
      4. can insert DNA into PLASMID into BACTERIA
    3. GEL is either AGAROSE or POLYACRYLAMIDE
      1. GEL is submersed in BUFFER
        BUFFER carries ELECTRICAL CURRENT
        WELLS at NEGATIVE end, DNA moves to POSITIVE end
        DNA is NEGATIVEly charged
        LARGER DNA fragments move SLOWER
        SMALLER DNA fragments move FASTER
        DNA visualized using fluorescent dye
        DNA is cut from GEL, PURIFIED, and used to RECOMBINE RNA
  3. enzymes that cleave DNA at different spots
    1. CLEAVE: break, cut
    2. enzyme cuts DNA at prescribed locations
      1. which INACTIVATES GENETIC INFO
  4. used by BACTERIA to fight off VIRUSES
  5. used in GENOME MAPPING
  6. 3 TYPES OF RESTRICTION ENDONUCLEASES
    1. type I and type III cleave w/ less precision, not used in manipulating DNA
    2. TYPE II
      1. recognizes specific DNA sequences
        most are PALINDROMES
        a PALINDROME is word/phrase that reads the same in forward or reverse
        DNA sequence is 4-12 bases
      2. results in STICKY or BLUNT ends
        STICKY ENDS are better
        insertion DNA also needs to have STICKY end, that is complementary
        can be joined
    3. EcoRI always cleaves the sequence 5-GAATTC-3
2. DNA LIGASE
  1. enzyme DNA LIGASE joins strands, FORMS phosphodiesters, FORMS back bone
  2. DNA ligase joins DNA fragments cut by restriction endonucleases and purified using an agarose gel
  3. DNA ligase also joins okazaki fragments on lagging strand in replication
DNA ---> DNA (REPLICATION) ---> RNA (TRANSCRIPTION) ---> PROTEIN (TRANSLATION)
1. RNA --> DNA
CHAPTER 18: GENOMICS
1. MAPPING GENOMES
  1. GENETIC MAP
    1. show relative location of GENES ON CHROMOSOMES, and they use genetic markers in order to do so, look at RELATIVE POSITION of markers
  2. PHYSICAL MAP
    1. actual DNA SEQUENCE ON GENOME, map of entire genome using markers, shows ABSOLUTE POSITION of markers
    2. 3 TYPES of PHYSICAL MAPS
      1. RESTRICTION MAPS
        CHROMOSOME MAPS
        use fluorescent stains/dye that produce patterns of bands on chromosome
        SEQUENCE TAGGED SITE MAPS (STS MAPS)
        uses unique short-stretches of genomic DNA, can be amplified by PCR, then analyzed, pieced back together
        useful for small genomes, genomes from organelles or viral genomes; NOT human genomes
        DNA cut w/ RESTRICTION ENZYMES (which cut at specific location)
        use ELECTROPHOREISIS to arrange DNA fragments by size
        PATTERN ANALYZED
        fragments put BACK TOGETHER based on SIZE and OVERLAP ---> CONTIG
        CONTIG is a contiguous segment of the genome
    3. SEQUENCING GENOMES
      1. ULTIMATE physical map is BASE-PAIR SEQUENCE of entire genome
      2. DIDEOXY TERMINATOR SEQUENCING
        use DIDEOXYNUCLEOTIDE CHAIN TERMINATORS
        there is NO HYDROXYL GROUP at 2 and 3 carbon in 5-carbon sugar
        another nucleotide can NOT BIND because of this
        this where DNA nucleotide ENDS
        A, T, C, and G tagged w/ different colored fluorescent DYE (each), fragments ANALYZED, fragments SEPARATED by length, detector READS sequence
      3. NEXT-GENERATION SEQUENCING (NGS)
        cost decreased significantly over time
        FEATURES:
        you can sequence DNA w/out constructing genomic library by CONVENTIONAL CLONING
        you can carry out MILLIONS of sequencing reactions at the SAME TIME
        also sequencing reaction can occur in solution; be read directly (NO ELECTROPHORESIS)
        CHALLENGES:
        produce LESS info
        read length is SHORT
        need MORE computing power
        ERROR PRONE if longer reads are done
2. THE HUMAN GENOME PROJECT
  1. originated in 1991
  2. GOAL: to sequence entire human genome
  3. uses shotgun sequencing
  4. CHARACTERIZING GENOMES
    1. found fewer genes than expected; expected 100,000, there is actually 20,000
      1. COMPLEXITY OF AN ORGANISM IS NOT NECESSARILY RELATED TO ITS GENE NUMBER OR GENOME SIZE
    2. PRODUCED REFERENCE SEQUENCE
      1. 1000 GENOME PROJECT
        looked at 1000 individuals from 26 populations, identified 80 million genetic variants
  5. BIOINFORMATICS
    1. computer programs to search for genes, and to assemble/compare genomes
      1. math; modeling
    2. COMPARATIVE GENOMICS
      1. compare genomes
      2. SYNTENY refers to conserved arrangements of DNA segments in related genomes
        relate function to other organisms
    3. PROTEOMICS
      1. PROTEOME: all of proteins that are produced from genome
      2. proteins are MORE DIFFICULT to study because...
        POSTTRANSLATIONAL MODIFICATIONS
        ALTERNATIVE SPLICING
        there are modifications that happen to proteins that do NOT reflect sequence of DNA
  6. GENOMICS CAN HELP TO IDENTIFY AND TREAT DISEASE
    1. identify genetic abnormalities
    2. identify victims by their remains
    3. tracing bacteria/viruses used in bioterrorism
    4. ETHICAL ISSUES
    5. GENE PATENTS
  7. THE HUMAN GENOME; THE GENOGRAPHIC JOURNEY
    1. DNA is able to trace back to earliest days of our species
      1. typos happen as DNA is passed on from generation to generation
        mtDNA; maternal; mitochondrial DNA comes from mother
        takes cells 8 HOURS to copy genome
    2. many ppl have AFRICAN ORIGIN
      1. recent common ancestor from 60,000 years ago
        patterns of human migration
    3. sampled 1,000 populations, over 500,000 participants

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