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NORTHEASTERN UNIVERSITY / Engineering / BIOL 2301 / How turner syndrome affects growth and sexual development?

How turner syndrome affects growth and sexual development?

How turner syndrome affects growth and sexual development?

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Study Guide 2


How turner syndrome affects growth and sexual development?



Chapter 5

∙ Human Chromosomes

o A chromosomes spread illustrating the human karyotype of 46 chromosomes 

o Geimsa straining gives characteristic bands on each  chromosome and allows for chromosome identification  ∙ Chromosome Painting

o Chromosomes can also be identified by chromosome  painting with specific probes

o How chromosome painting works 

 Repetitive DNA sequences have been identified on  each chromosome  

 DNA complementary to these sequences is  

synthesized and labeled with a specific dye

∙ These DNAs are called probes 

 Probes for each chromosome are labeled with a  

specific color dye  

 Probes are allowed to hybridize to a prep of  

metaphase chromosomes  

 A microscope is used to take a photo of the labeled  chromosomes  

 Computer aids in alignment of matching  


How chromosome painting works?



chromosomes  

∙ Dosage Compensation

o Dosage compensation adjusts for differences in the  numbers of sex chromosomes  

o In many mammals, dosage compensation is accomplished  by X inactivation in females

o The inactivated X is called a barr body 

o The inactive X is not completely inactive – one of the genes still expressed is Xist which acts to keep the X chromosome in its inactive state  

o Most genes in the inactive X are not expressed

∙ Genes Present on both X and Y allow X-Y  


A chromosomes spread illustrating the human karyotype of, how many chromosomes?



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o The tips of the X chromosome escape inactivation because  these regions contain genes that are also on the Y  

chromosome  

o Regions of X-Y homology are called pseudoautosomal  regions 

o Patterns of inheritance of genes in pseudoautosomal  regions are indistinguishable from autosomal inheritance  patters  

o SRY is the master sex determination gene 

 Presence of the gene SRY determines male  

development  

 SRY encodes a transcription factor called testis

determining factor (TDF)

 Genes turned on by TDF result in development of  testis and other male characteristics  

 Mutations in SRY can result in XY females, and  

recombination errors that transfer SRY to X can result in XX females with male characteristics  

o Turner Syndrome  

 Turner syndrome individuals are missing one of their  X chromosomes  

 Turner syndrome affects growth and sexual  

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 Girls with this disorder have a variety of symptoms  including a short, stocky stature, delayed puberty,  

neck and facial abnormalities, and infertility  

o Kleinfelter Syndrome

 Kleinfelter syndrome individuals are XXY

 Kleinfelter syndrome affects sexual development   Males with this disorder have a variety of symptoms  including little body hair, breast development, and  Don't forget about the age old question of sebastien robidoux

infertility

o The Y chromosome can be used to trace the history of  human populations  

 Most of the Y chromosome does not undergo  

recombination – therefore Y is passed down mostly  unchanged from father to son 

 Haplotype – set of alleles at two or more loci in a  particular chromosome (usually 20-30 alleles are  

used to define a particular Y chromosome)  

(Alternative Definition: a “chromosome type” defined by multiple alleles)

∙ A haplotype is a combination of alleles  

(sequence variants) at multiple loci that are  

transmitted together on the same chromosome

∙ Alleles that are linked (nearby each other) tend

to be inherited together  

∙ New haplotypes are slowly produced by rare  

recombination or mutation events  

 SSR polymorphisms are usually used to trace the  inheritance patterns of Y chromosomes

 Men with similar Y haplotypes have a recent common ancestor  

∙ The more similar the Y, the more recent the  

common ancestor  

∙ Chromosome Abnormalities We also discuss several other topics like what is a nonpenetrating (np) solute?
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o Human individuals with chromosome abnormalities do not  usually survive to birth

o In about half of all spontaneous miscarriages, the fetus has a major chromosome abnormality  

o Trisomy – a third copy of one of the chromosomes o Aneuploidy – not having the same number of each  chromosome

o Polyploidy – an extra set (or 2) of all of the chromosomes   Only seen in plants

o Monosomy – only one of a particular chromosome  ∙ Trisomy Disorders

o Down Syndrome is the result of trisomy 21 (three copies of  chromosome 21)

 Usually, trisomy 21 is the result of chromosome 21  nondisjunction  

o Trisomic chromosomes undergo abnormal segregation Don't forget about the age old question of drawn below is the structure of crestor® (rosuvastatin), a medication used to reduce cholesterol. assign the absolute configurations of the two chiral centers as r or s.

∙ Chromosomal Deletions  

o A chromosome with a deletion or deficiency is missing  genes  

o Deletions can be mapped because wild type copies of the  gene will be missing – recessive phenotypes will show up  (be uncovered) in the heterozygote  

o Mapping a deletion using a test cross  

∙ Chromosomal Duplications  

o A chromosome with a duplication has extra copies of a  gene or genes  

o Duplications are caused by unequal crossover  

∙ DNA breaks can result in inversions  

o Chromosomes with inversions are fine in mitotic cell  divisions, but cause problems in meiosis 

o Inverted regions of chromosomes form loops during gene  pairing (synapsis) in meiosis

o Crossing over within the inversion loop results in acentric  and dicentric chromatids  

∙ Chromosomes and Centromeres

o Having just one centromere is required for correct  chromosome segregation 

o Acentric chromosomes lack centromeres and are  genetically unstable

o Dicentric chromosomes are also unstable – if the two  centromeres attach to opposite poles, the chromosomes  can be stretched and get broken  

∙ Translocations

o Translocations are chromosomal abnormalities which occur  when chromosomes break and the fragments rejoin to  other chromosomes  

o Reciprocal Translocations  

 In a reciprocal translocation, two non-homologous  chromosomes break and exchange fragments

o Robertsonian translocation is a type of “centric fusion”  

translocation  

 Robertsonian translocations are caused by fusions of  

acrocentric chromosomes 

∙ Polyploidy

o Polyploid species have multiple sets of chromosomes  

o Polyploidy is only found in plants 

∙ Monoploid vs. Haploid  

o Monoploid – one of each chromosome

o Haploid – half of the total number of chromosomes  

o In a diploid organism, monoploid = haploid  

o In a tetraploid (36) organism, the haploid (half) count is 18  and the monoploid count is 9

∙ Major Concepts 

o Each human chromosome can be individually identified by  staining methods  

o Dosage compensation adjusts for the fact that males only  have one X chromosome, and females have two  

o Y chromosome haplotypes allow tracking of human  

populations  

o Chromosomal abnormalities have a variety of severe  

consequences  

Chapter 6  

∙ Chemical Structure of DNA  

o DNA is composed of a sugar deoxyribose phosphoric acid  and 4 nitrogen-containing bases 

o Purine Bases (two rings)

 Adenine (A)

 Guanine (G)

o Pyrimidine Bases (one ring)

 Cytosine (C)

 Thymine (T)

 Uracil (U) – found in RNA

∙ Nucleosides and Nucleotides

∙ Polarity of DNA  

o Each DNA strand has a polarity or a directionality 

o The 5’ phosphate is chemically linked to the 3’ OH of the  next nucleotide  

o Antiparallel – the arrangement of the two strands in a  double helix  

∙ Bases pair through hydrogen bonding 

o Hydrogen bonds – weak bonds in which F, O, or N share a  hydrogen atom  

o A – T base pairs form two hydrogen bonds

o G – C base pairs form three hydrogen bonds  

o The 5’ phosphate is chemically linked to the 3’ OH of the  next nucleotide  

∙ DNA Replication

o Semi-conservative replication – each DNA strand is used as a template for a new strand  

o Template – the older strands that are used to make a copy  (parental strands)  

o DNA polymerase extends the DNA strand 5’ to 3’ by adding base-pairing nucleotides

o DNA polymerase can proofread (fix) its mistakes  This error correcting mechanism significantly  decreases the rate of mutations caused by mistakes  during DNA replication  

o Bi-directional Replication of DNA  

 Replication origin – DNA sequence where replication  begins  

 Replication fork – region where parental strands are  separated and new strands are being synthesized   Replication initiation – the process of starting a new  replication fork

o Eukaryotic DNA molecules contain multiple origins of  replication 

o DNA synthesis begins with an RNA primer 

 Primer – a short stretch of RNA that provides a 3’ OH  group for DNA polymerase to use to start adding new DNA  

 RNA primers are later removed and replaced with  DNA

 DNA (and RNA) synthesis always occurs in a 5’ to 3’  direction 

∙ This means the STRAND grows 5’ to 3’

∙ DNA sequencing methods are based on DNA replication  o Sanger Sequencing (Original)

o “Next Generation” Sequencing  

 These newer techniques greatly increase throughput  ∙ They generate more bases of sequence pre  

time and money spent compared to Sanger  

sequencing  

∙ The length of any individual read is often less  

than seen with Sanger  

 The two most common techniques are Illumina  

sequencing and 454 Pyrosequencing  

o PCR, the polymerase chain reaction, based on DNA  replication, is a technique for making lots of copies of DNA  in a tube  

 PCR allows amplification of a specific segment of  DNA from a very small amount of sample 

 PCR reaction includes:

∙ DNA template

∙ Primers

∙ DNA polymerase

∙ Buffer (adjusts pH and provides Mg++)

∙ dATP, dTTP, dCTP, dGTP

 Heat-tolerant DNA polymerase for PCR

∙ Human enzymes work best at 37 C

∙ PCR uses a DNA polymerase that can tolerate  

high temperatures

∙ These polymerases are isolated from archaea  

that populate hot thermal vents  

 Each cycle of PCR has three steps 

o The DNA polymerase synthesizes DNA 5’ to 3’ beginning  with the primer  

 The primers tell the polymerase where to start  

synthesizing the DNA  

o PCR results in an exponential amplification of DNA

o “Real Time” PCR tracks the amplification of the DNA as it  occurs by monitoring fluorescence  

o SYBR is a dye that is fluorescent when incorporated into a  double stranded DNA  

o In “real time” PCR, the machine takes a fluorescent image  each cycle  

∙ Major Concepts 

o DNA strands have a chemical polarity with a 3’ OH at one  end and a 5’ phosphate at the other  

 Double stranded DNA is antiparallel

o A new DNA strand is copied from each parental strand by  the sequential addition of nucleotides by DNA polymerase  o DNA replication requires many different enzymes, but the  actual replication of DNA is performed by DNA polymerase, which catalyzes a phosphodiester bond between the 5’  phosphate and the 3’ hydroxyl residues

o DNA sequencing and PCR are based on DNA replication  Chapter 7

∙ Basic microbiology techniques – bacterial culture  o Some types of bacteria can easily be propagated in the lab  for study  

o Solid media (plates) and liquid media (flasks or tubes) are  used  

o Colonies contain millions of cells  

o Growth media contains amino acids and sugars that  support bacterial growth (plates also contain agar – a solid, jello-like support substance)

o Bacterial growth is exponential, until nutrients run out  o Calculating cell concentration by serial dilution 

 By this serial dilution method, the original culture has been diluted one million (106) fold  

 To determine how many cells were in the original  culture, grow 0.1 ml of the diluted culture, then  

multiple the number of colonies by the reciprocal of  the plating dilution factor  

 Ideally, count colonies on a plate with between 30  and 300 colonies

 Failing that, count the plate that comes closest to  that range

o Another way to Count Cells (Need Special Equipment)  Petroff-Hauser slide counter; similar to a  

hemocytometer, but with less depth in the field of  

view  

 1. Dilute the cells  

 2. Pipet 1 ul diluted cells into the slide counter  

 3. Count the average number of cells in the squares,  multiple by volume of square  

 4. Multiply by your dilution factor to get cells/ml in  the original solution  

 This way doesn’t tell you whether or not the cells are  viable

∙ Genetic analysis in bacteria uses three principle types of mutant o 1. Antibiotic resistant mutants – mutation allows growth in  the presence of an antibiotic  

 Example: Cells that can grow in the presence of  

ampicillin (ampR)

o 2. Nutritional mutants (auxotrophs) – cells that can’t grow  without a nutrient supplied by the researcher (such as an  amino acid)

 Example: Cells that can’t synthesize methionine, an  amino acid, and therefore require it in the media  

(met-)

o 3. Carbon-source mutants – cells that can only metabolize  certain sugars (carbon sources)

 Example: Cells that grow fine on glucose but can’t  metabolize lactose (lac-)

∙ Antibiotic Resistance

∙ Persister cells are also a serious problem

o Persister cells are dormant cells that are not killed by most  antibiotics that target dividing cells

o Persister cells are NOT mutant (resistant) cells, they are  just regular bacteria that are hanging out until conditions  improve  

o Once the antibiotics are removed, the surviving cells can  re-establish the infection  

∙ Ways by which bacteria acquire antibiotic resistance  

∙ Bacterial Chromosomes  

o E.coli has one circular chromosome that contains the  majority of the genome  

 Haploid

 Reproduces asexually  

 Genetics are through extra DNA elements  

o Bacteria share genes by transferring pieces of DNA  Plasmids are small, usually non-essential, DNA  

molecules maintained in bacterial cells  

 Plasmids confer traits like antibiotic resistance and  can be shared from cell to cell, even from species to  species  

 Plasmids are much smaller than the bacterial  

chromosome  

∙ Transformation of Bacteria

o Transformation is the process by which bacteria pick up  DNA from their environment  

o The DNA may come from a variety of sources, but most  likely it is the remnants of DNA from dead bacterial cells o The plasmid contains an antibiotic resistance gene so that  cells that have taken up the plasmid can be selected

o Some plasmids contain genes that enable plasmid DNA to  be transferred between cells by conjugation  

 Conjugation – joining of bacterial cells in order to  transfer DNA  

 Conjugative Plasmids – plasmids that can be  transferred this way  

 Most small plasmids are non-conjugative but can use  recombination to hitch a ride with conjugative  

plasmids  

o The “F factor” is a conjugative plasmid 

 F factor containing E. coli cells are designated F+  cells, cells without F are called F-

 F factor is 100 kb in length and encodes many genes  that ensure its own maintenance in the cell and  transfer from cell to cell  

 The F factor directs the formation of the F pilus   Step 1: Make the pilus  

 Step 2: Replicate the F factor plasmid and transfer it  simultaneously

o Transfer of F factor from cell to cell works by rolling circle  replication 

 Circular DNA is nicked by a nuclease  

 New nucleotides are added, displacing the other  strand  

 There isn’t an “end” to be reached in rolling circle  DNA replication, so lots of copies of F-factor can be  made  

o F Factor Conjugation 

 Transfer of F factor results in both cells containing F  factor  

 Transfer of F factor only takes a few minutes

 In laboratory conditions, F factor can quickly spread  from a few donor cells through the whole population   In nature, conjugation and transfer of F factor is  balanced by selection against it  

∙ Only 10% of E. coli in nature contain F factor  

∙ Transposable Elements  

o Transposable elements are DNA sequences that can jump  from one position to another or from one DNA molecule to  another

o The smallest and simplest are insertion sequences, which  are 1-3 kb in length and encode transposase  

 Larger ones encode multiple genes and are called  transposons  

o Transposase is the enzyme that excises transposable  elements and integrates them back in somewhere else  o Structure of transposable elements in E. coli  

 Tn5 encodes resistance genes to three different  antibiotic resistance genes  

 When Tn5 inserts itself into a conjugative plasmid it  is widely disseminated from bacteria to bacteria

o Recombination between transposable element DNA  sequences allows non-conjunctive plasmids and pieces of  genomic DNA to be transferred to conjugative plasmids 

o The recombined plasmid is called a cointegrate 

o Recombination can also “release” the small plasmid from  the conjugative plasmid  

o Enzymes that catalyze recombination are called  recombinases 

∙ Transduction

o A phage (or bacteriophage) is a virus that infects bacteria o DNA, including pieces of bacterial DNA, can be transferred  between bacterial cells by a transducing phage

o Generalized transducing phage transfer DNA bits from  bacteria by mistake – instead of phage DNA genome  o Specialized transducing phage produces phage that  contain both phage and specific bacterial genes  

o Phages are viruses that infect bacteria 

o Phage P1 transfers bacterial DNA  

∙ The Lytic Cycle

o The lytic cycle – reproductive cycle of a phage

o Phage DNA enter a cell and replicates repeatedly  o The bacterium is completely taken over and converted to  phage production

o Newly formed phages break out (lyse) the bacterial cell  and infect other bacteria

∙ Phage titer (plaque forming units) can be determined by serial  dilution 

o 1. Dilute the solution of phage

o 2. Add the diluted phage to a lawn of bacteria

o 3. Count the plaques

o 4. Multiply by the dilution factor  

∙ The lysogenic cycle  

o The lysogenic cycle – no progeny particles are produced o Phage DNA enters a cell and integrates itself into the  bacterial genome

 Inserted DNA is called a prophage 

 The surviving bacterial cell is called a lysogen 

o Lysogenic phages are called temperate phages 

o Lytic phages are called virulent phages 

o Prophages can be activated, excise themselves from the  genome, and begin the lytic phase 

o Many other types of virus (for example herpes virus) also  have lytic and lysogenic life cycles  

o The phage inserts its genomic DNA into the bacterium, but  doesn’t start immediately producing more phage virus  particles  

o Instead, the phage DNA becomes integrated into the  bacterial chromosome  

o The phage DNA is then replicated by the bacterium along  with the host DNA  

o When conditions are rights, the phage DNA will use its own  recombinase to pop out of the bacterial chromosome and  enter the lytic phase, producing many phage and killing  the bacterial host

∙ Bacteria use restriction enzyme as a defense against phage  ∙ Key Terminology 

o Transformation – uptake of DNA from a source outside of  the cell

o Conjugation – physical joining of two bacterial cells and  transfer of DNA  

o Transduction – transfer of bacterial DNA via a phage (virus) o Conjugative plasmid – a circular piece of DNA transferred  by conjugation (for example, F factor)

o Transposable elements – DNA sequences that can “jump”  from one position to another in the genome  

o Phage – a virus that infects bacteria

o Transducing phage – a virus that transfers bits of bacterial  DNA along with its own viral genome  

o Lytic of virulent phages – viral life cycle stage including  replication of virus and cell lysis  

o Lysogenic or temperate phages – viral life cycle stage in  which the virus integrates its DNA into the host genome ∙ Major Concepts 

o Bacteria can take up foreign DNA by transformation  o Bacterial DNA can be transferred by conjugation or by  transduction by bacteriophage  

o Transfer of DNA elements is a major source of antibiotic  resistance and virulence in bacteria  

o Bacteriophages have two life cycles, the lytic cycle and the lysogenic cycle  

o Restriction enzymes produced by bacteria cut DNA at  specific sequences and are very important research tools  

Chapter 8  

∙ Gene Expression

o Gene expression – using the information stored in DNA to  produce RNA and protein molecules that determine the  phenotype or organisms  

∙ Prokaryotic Gene Structure 

∙ Transcription  

o Transcription – the synthesis of an RNA molecule from a  DNA template  

∙ Chemical Structure of RNA  

o RNA is similar to DNA: the only differences are in the sugar  in the backbone (ribose instead of deoxyribose), and RNA  does not have T (thymine) it has U (uracil) instead 

o The precursors in RNA synthesis are A, C, G, and U  ∙ Steps in transcription of an RNA molecule 

o 1. Promoter recognition (find the beginning of the gene) o 2. Chain initiation (start transcribing)

o 3. Chain elongation (make the RNA molecule)

o 4. Chain termination (stop at the right place)

∙ Promoter Recognition  

o E. Coli  

 In E. coli, RNA polymerase identifies promoters by  binding consensus sequences at -10 and -35 bp  

 Transcription starts at a position approximately 10  nucleotides after the consensus sequence  

o Eukaryotic promoter regions are longer and more complex and variable than prokaryotic promoters  

∙ Transcription Initiation 

o RNA polymerase binds the promoter  

o DNA separates

o The first RNA nucleotide base pairs with the starting base  on the DNA

o A second RNA nucleotide enters, and RNA polymerase  forms a bond between its 5’ phosphate and the 3’ hydroxyl of the first nucleotide  

∙ Sequence of bases in an RNA molecule is determined by the  sequence in the DNA template 

o Each RNA base is chosen for its ability to base pair with the DNA template strand  

∙ RNA polymerase forms a bond between the 3’ OH group at the  end of the chain and the 5’ phosphate group of the next  nucleotide 

o RNA molecule elongates in the 5’ to 3’ direction 

∙ Transcription Termination  

o This stem-loop, or hairpin, in the mRNA signals to RNA  polymerase (which has already passed), to stop  

transcription

o Transcription does not have to be complete before a new  transcript is initiated

∙ Transcription  

o Some genes are transcribed from one DNA strand as a  template  

o Others are transcribed from the other DNA strand o The direction of transcription (left or right) is determined  by the promoter at the beginning of each gene  

o Genes are always transcribed from 5’ to 3’  

∙ Transcribed RNA molecule  

o In prokaryotes, the primary transcript is the messenger  RNA 

o In eukaryotes, the primary transcript undergoes further  modifications (RNA processing) before it becomes mRNA ∙ Eukaryotic gene structure

∙ Eukaryotes only

o RNA processing 5’ cap 

 The 5’ end is altered by the addition of a modified  guanosine residue (7-methyl guanosine) called the  cap 

 The 5’ cap is necessary for the ribosome to bind the  mRNA and begin protein synthesis in eukaryotes  

o RNA processing poly A tail 

 The 3’ end is modified by the addition of a sequence  called the poly A tail 

 The poly A tail can be up to 200 A residues long, and  is thought to regulate RNA stability  

∙ RNA Splicing  

o Introns – intervening sequences that are removed by RNA  splicing  

o Exons – the coding segments of the gene  

 Exons are joined to form the coding sequence  

o The 2’ OH group of an Adenine forms a bond with the  phosphate group at the 5’ splice site, creating a loop in the RNA molecule  

o The released free 3’ OH end of the exon sequence then  reacts with the start of the next exon sequence, joining the two exons together  

o The intron lariat (lasso) is released

 The exons are joined together

o Splicing is usually controlled by a complex of proteins and  RNAs called the spliceosome

o Self Splicing Introns

 Self-splicing introns fold into structures that bring the two nucleotides that engage in the splicing reaction  into proximity  

 RNAs that fold up and have enzymatic activity on  their own (catalyze reactions) are called ribozymes 

∙ Primary transcripts of many genes are alternatively spliced to  yield different products  

o The same transcript (mRNA) can be processed differently  in different cell types, or in different conditions  

o Different proteins can be produced from the same gene  transcript by inclusion or exclusion of specific exons  o Alternative splicing is very common

o When primary transcripts are alternatively spliced,  different proteins are produced  

∙ The new RNA is modified as it is being transcribed  

∙ Review of the characteristics of mature mRNAs ready for  translation

∙ Gene Expression

o Gene expression – using the information stored in DNA to  produce RNA and protein molecules that determine the  phenotype of organisms

o Translation – using the information in the mRNA to produce  a protein  

∙ Proteins

o Proteins carry out most of the functions of cells including  catalyzing biochemical reactions, regulating gene  

expression, and providing mechanical structure 

o Proteins are composed of one or more polypeptide chains o Polypeptide chains are composed of amino acids linked  together by peptide bonds  

∙ General Structure of an Amino Acid  

∙ Peptide Bonds  

o A polypeptide chain  

 Polypeptides are numbered in the order they are  

added to the chain during synthesis, from N to C  

 This order from N to C is collinear (in the same order) as the mRNA 5’ to 3’

∙ In prokaryotes, transcription and translation are coupled ∙ In eukaryotes, transcription and RNA processing occur in the  nucleus, and translation occurs in the cytoplasm 

∙ Steps of Translation 

o 1. Initiation 

 Initiation factors bind the mRNA. The ribosome  

assembles. Initiation factors help start translation at  the start codon  

o 2. Elongation 

 A charged tRNA enters the A site of the ribosome. A  peptide bond forms between the amino acid on the  tRNA and the growing polypeptide chain. This tRNA  then moves to the P site of the ribosome. The  

ribosome moves to the next codon and waits for the  next charged tRNA  

o 3. Termination 

 When a stop codon is reached release factors help  the ribosome dissociate  

∙ Components of the Translation System

o mRNA – provides the coding sequence of bases that  determines the amino acid sequence and brings ribosomal  subunits together

o ribosome – the protein and RNA machine that directs  protein synthesis  

o tRNA – transfer RNA molecules bring amino acids to the  ribosome and put them in the correct order by binding to  codons in the mRNA  

o aminoacyl-tRNA synthetase – catalyzes the attachment of  a particular amino acid to its corresponding tRNA

o Initiation, elongation, and termination factors – control the  synthesis of the polypeptide chains  

∙ Initiation

∙ Translation  

∙ The Shine-Dalgarno Sequence  

o This sequence helps prokaryotes (bacteria, actually) recruit the ribosome to the mRNA to initiate protein synthesis by  aligning the ribosome with the start codon  

∙ The Kozak Sequence  

o Similar to the Shine-Dalgarno sequence, the Kozak  sequence helps the ribosome identify the start codon in  eukaryotes

o In eukaryotes, the ribosome binds the 5’ cap and then  scans down the mRNA to find the Kozak sequence near the  AUG  

o When researchers want to express proteins using a plasmid they often need to include a Kozak sequence so that  translation starts at the right spot  

∙ Elongation 

o 1. A new charged tRNA inters the A site and binds to the  codon by complementary base pairing  

o 2. A new peptide bond is formed between the amino acid in the P site and the amino acid in the A site to elongate the  polypeptide  

o 3. The ribosome moves along to the next codon on the  mRNA, and the cycle repeats

∙ A charged tRNA is a tRNA molecule attached covalently to an  amino acid  

∙ Elongation  

∙ Termination

∙ Reading codons using the genetic code  

o The code is read three nucleotides at a time from a fixed  point  

 This is referred to as a reading frame 

∙ Open Reading Frames

o An mRNA could have many places with an AUG, resulting in many possible reading frames  

o The reading frame that starts with an AUG and continues  without any stop codons to the end of the RNA is called the open reading frame 

o Open reading frame (ORF for short), predictions are based  on DNA sequence  

 The researcher just finds the longest stretch with no  stops  

∙ Frameshift Mutations

o Mutations that add or delete a base pair (or two) are called  frameshift mutations because they shift the reading frame  ∙ Some tRNA anticodons can pair with more than one codon

o The third base in the anticodon is less spatially  

constrained, and therefore can “wobble” (tolerate  

mispairing)

o Some tRNA anticodons include a 5th base: inosine Pro ∙ Protein folding is the process by which a linear polypeptide chain  becomes a 3D protein  

o The final shape of the protein is determined by the  chemistry of the amino acid side chains 

o Correct folding is very important – diseases such as  Huntington’s and Alzheimer’s are caused by misfolded,  aggregated proteins  

o Most proteins fold correctly as they leave the ribosome  o Other proteins are helped to fold by heat shock proteins  and by the barrel-like chaperonin complex  

∙ Proteins are often composed of more than one polypeptide ∙ Major Concepts 

o RNA is similar chemically to DNA, but is composed of A, C,  G, and U (instead of T), and contains ribose (instead of  deoxyribose) sugars

o Transcription proceeds 5’ to 3’ and produces an RNA  transcript  

o Genes include 5’ regulatory regions (including the  promoter), the coding region, and the 3’ regulatory regions o In eukaryotes, the primary transcript is modified with a 5’  cap, a 3’ poly A tail, and is spliced to remove introns  o Transcription and RNA processing are coordinated  processes involving molecular machinery consisting of  protein and RNA molecules  

o Polypeptide chains are linear polymers of amino acids o Translation takes place on the ribosome and requires the  mRNA, tRNA, ribosomal proteins and RNAs, and various  accessory factors

o Proteins are synthesized from N to C. This is collinear with  the 5’ to 3’ mRNA and the 5’ to 3’ coding strand of the DNA o The genetic code is read as triplets. The position of the  start codon (ATG) determines the reading frame  

o A 3 bp region of the tRNA, the anticodon, is responsible for  “matching” the right amino acid to the codon on the mRNA

Chapter 9

∙ Gene Regulation

o Gene regulation – how genes are turned off and on to  produce specific cell types and to respond to changing  environmental conditions  

o Gene regulation is necessary for organisms to respond to  changing environmental conditions  

∙ In prokaryotes, adjacent genes are often transcribed as a single  unit  

o Polycistronic mRNAs undergo coordinate gene regulation  o Coordinate gene regulation means that all of the genes are transcriptionally regulated together  

 In other words, turned “on” or “off” as a unit  

o Polycistronic mRNA - a set of genes transcribed together  from one promoter is called an operon  

o A transcriptional activator can bind and turn gene  expression “on” 

 In positive regulation, the default state of  

transcription is “off”, unless an activator turns it “on”

o In positive feedback, the protein product of a gene turns on its own transcription

 In positive feedback, the protein that is expressed  turns its own expression ON

 Positive feedback amplifies a weak original signal 

o A transcriptional repressor can bind and turn gene  expression “off”

o Transcriptional Regulation In Prokaryotes  

 In bacteria on-off gene activity is often controlled  through transcription 

 “OFF” actually means a low basal level of  

transcription – a small amount of gene is almost  

always produced  

 “ON” genes also have a wide range of expression  Gene regulation is more like a dimmer switch than an on/off switch, although the on/off terminology  

remains common  

∙ E. coli lactose metabolism  

o Lactose metabolism requires two proteins; an enzyme, β galactosidase and a transporter, lactose permease  

o The lac operon – a series of genetic mutation studies  identified genetic elements in the lac operon – one of the  first examples of gene regulation  

 i encodes a protein (the repressor) that inhibits  

transcription of the operon

 p is the DNA element (the promoter) that RNA  

polymerase binds to  

 o is a DNA element (the operator) that lacl (the  

repressor) binds to  

 z, y, and a are genes that encode proteins for sugar  metabolism  

o On-Off gene regulation in the lac system  

 If no lactose is present, β gal and lactose  

permease are not made by the cell 

 When lactose is present, β gal and lactose  

permease are quickly synthesized

 When lactose is removed, β gal and lactose  

permease mRNA decreases rapidly, but the proteins  are stable and last quite some time  

 These observations led to the theory that  

transcription of the lactose genes is inducible, and  

that the inducer is lactose  

o Lacl is a regulatory gene that produces a repressor protein  that keeps the system “off” by binding to the lacO operator and blocking transcription

o In the presence of lactose, Lacl does not bind DNA 

(lactose)

o In the presence of lactose, the lac operon is transcribed  and translated 

o The lac operon is also subject to regulation by glucose   If both glucose and lactose are present in the culture  medium, transcription of the lac operon is shut down until all the glucose has been consumed  

o Effect of glucose on the lac operon is indirect

 If glucose is low, or bacteria are otherwise starved,  the cAMP levels are high 

∙ cAMP is a signaling molecule

∙ Inducible Transcription

o An inducer can bind and remove the repressor, thereby  turning gene expression on

o Inducers are often small molecules that bind the repressor  and cause it to lose DNA binding activity  

∙ CRP and adenyl cyclase are required for transcription of the lac  operon 

o Adenyl cyclase is the enzyme that makes cyclic AMP o CRP – cyclic AMP receptor protein

o 1. CRP binds cyclic AMP

o 2. CRP binds lac promoter and positively regulates (turns  on) transcription of the lac operon  

∙ The lac operon is ON only when cAMP-CRP is present (low  glucose) AND the repressor is absent (presence of lactose) ∙ The tryptophan (trp) operon  

o The trp operon quickly responds to the amount of  tryptophan in the cell by dialing up or down the amount of  transcription of the enzymes needed to make tryptophan  ∙ Features of the trp operon  

o When adequate tryptophan is available, gene expression is repressed 

o The trp operon encodes a set of biosynthetic enzymes, all  necessary to synthesize tryptophan  

o The trp operon is “tunable”  

 The transcription level is adjustable based on the  concentration of tryptophan in the cell  

 The regulatory protein is encoded by trpR, which is  not encoded in the trp operon  

∙ Repressible Transcription

o Gene transcription is on until the active repressor binds o Aporepressor – a protein that has no DNA binding activity  on its own, but which requires the presence of a co repressor to turn transcription off  

o Many biosynthetic (anabolic) pathways are shut off by the  final substrate in the pathway  

 Presence of the final product signals to the cell that  the enzymes aren’t needed any more

∙ Regulation of the trp operon 

∙ The leader sequence (trpL) acts like a dimmer switch to tune trp  operon expression 

o The trpL transcript (mRNA) can “sense” the level of  tryptophan  

o In prokaryotes, the ribosome can hop on the transcript and  start translating it right away – it doesn’t wait for RNA  polymerase to finish transcribing the message  

o This feature allows the coupling of translation effects with  transcription effects  

o This method of controlling transcription is called  attenuation 

∙ trpL  

o trpL codes for a short 14 aa peptide

o There are two consecutive trp codons that can be used to  measure the tryptophan supply in the cell, through  translational coupling  

o High tryptophan means trp-tRNA will be available and trpL  will be translated without problem 

o Low tryptophan means trp-tRNA will be available and trpL  will be translated without problem 

∙ Transcription Termination

o This stem-loop, or hairpin, in the mRNA signals to RNA  polymerase to stop transcription

∙ Chain termination of trpL  

o If a ribosome stalls on the truptophan codons while waiting for trp-tRNAs, a particular stem-loop, called the anti terminator, will fold in the mRNA, and RNA polymerase will  continue to transcribe the trp genes  

o If trp-tRNAs are abundant, the ribosome will move farther  down the message, allowing the terminator stem-loop to  form, which will cause RNA polymerase to stop transcribing the trp synthesis genes  

∙ Anti-Terminator prevents the terminator from forming

∙ The terminator looks like a transcription termination site – when  RNA polymerase sense a terminator (in the mRNA) it will stop  transcribing  

∙ When tryptophan is plentiful, trp operon genes E, D, and C are  not need 

o 1. A ribosome follows right behind RNA polymerase,  translating the trpL transcript until it is halted by a stop  codon  

o 2. When the ribosome stops at the stop codon, a  terminator stem-loop forms in the transcript (this is the  normal end of trpL)

o 3. RNA polymerase sense the terminator (in the mRNA) and dissociates – no transcription of E, D, or C

∙ When tryptophan starts to get low, the trp operon genes E, D, C  are needed 

o 1. The ribosome stalls and waits at the two tryptophan  codons

o 2. This permits formation of the anti-terminator  

o 3. As the ribosome waits around for the truyptophan tRNA  to come, RNA polymerase gets ahead and transcribes the  trpE, D, and C genes  

∙ Attenuation is for prokaryotes only 

o In eukaryotes, transcription takes place in the nucleus and  translation takes place in the cytoplasm  

o Therefore, ribosomes don’t bind the RNA while it is still  being transcribed

∙ Cells in a multicellular organism have many proteins in common  o Many processes are common to all cells, and any two cells  in a single organism therefore have many proteins in  common 

o For example, RNA polymerases, DNA repair enzymes,  ribosomal proteins, enzymes involved in the central

reactions of metabolism, and many of the proteins that  form the cytoskeleton are needed in all cells

o Many of these common proteins are called housekeeping  genes 

∙ Gene Expression

o Gene expression differences generate different cell types in multicellular organisms

o Differences in gene expression lead to the differences in  size, shape, and function

o One genome leads to multiple cell types 

 Each cell has the same DNA but different genes are  expressed  

o Different gene expression means different genes are  transcribed and translated 

 The amount of transcription of a given gene will also  vary by cell type and by environmental condition  

o Gene expression can be regulated at many steps along the  way from DNA to RNA protein

∙ Regulation of Transcription in Eukaryotes  

o The transcription complex in an aggregate of protein  factors that combines with the promoter to initiate  

transcription 

o The basal transcription factors are proteins in the complex  that are used in the transcription of many different genes  o Transcription activation occurs by a mechanism called  recruitment – the interaction of transcription factors with  promoter and enhancers  

o Enhancers are usually in the general vicinity of the gene  they regulate, although they can be anywhere: 5’, in  introns, or 3’ of the gene

∙ Transcription factors bind DNA to regulate gene expression o Transcription factors can activate or repress transcription  by binding to DNA sequences 

o Types of Transcription Factors

 General (or basal) transcription factors 

∙ Bind DNA near the promoter and associate with

RNA polymerase  

∙ Required for low-level basal transcription  

 Transcriptional activators and repressors 

∙ Bind enhancer DNA sequences

∙ Activate or repress transcription of specific  

genes

∙ Required for the cell to change gene expression

in response to changing conditions  

o Structure of Transcription Factor  

 DNA binding domain (DBD) which binds specific  

sequences of DNA (enhancer or promoter sequences) adjacent to regulated genes  

∙ DNA sequences which bind transcription  

factors are often referred to as response  

elements 

 Activation domain (AD) regulates transcription by  interacting with other proteins (such as basal  

transcription factors or RNA polymerase)  

∙ The helix-turn-helix motif is a protein fold commonly found in  transcription factors that bind DNA  

o The helix-turn-helix motif has pairs of helices separated by  a linear chain of amino acids  

o This structure fits well into the DNA major groove ∙ The zinc finger motif is also commonly found in transcription  factors

o Folded structure incorporates a zinc ion

o These proteins preferentially bind specific DNA sequences

∙ Expression of a “master” transcription factor can turn on a set of  downstream transcription factors (FLY EYE EXAMPLE) o Transcription factors use feedback loops to make sure the  “make an eye” program stays ON  

o Transcription factors turn on the genes that produce  proteins needed to construct an eye  

o Eye development results 

o The gene eyeless is a transcription factor that turns on eye  development  

∙ Cells change the genes they express in response to external  signals like nutrient availability (GALACTOSE UTILIZATION  EXAMPLE)

o When the sugar galactose is around, yeast turn on the  enzymes needed to metabolize it  

o If galactose is not around, the transcription of these  enzymes remains off  

∙ GAL4

o GAL4 binds DNA using a zinc finger  

o The DNA binding domain binds DNA and the activation  domain attracts RNA polymerase 

o The DNA sequence bound by GAL4 is called a UAS  (upstream activator sequence) – the same as an enhancer  ∙ Galactose metabolism requires the transcription and translation  of several enzymes 

o These genes are close together, but they are not in an  operon  

o Each gene has its own promoter and makes its own mRNA o These genes are synthesized (induced) only when  galactose is present, and can all be turned on by GAL4  ∙ Regulation of transcription of GAL genes by GAL80, GAL3, and  GAL4

o When there is no galactose around, GAL80 binds GAL4 and  prevents transcription of the galactose metabolizing  enzymes

∙ Comparison of regulation of yeast Gal genes and E. coli lac genes 

∙ Activating transcription by recruiting RNA polymerase  o Transcriptional activation requires the activating  transcription factor, the basal transcription factors, and the RNA polymerase holoenzyme 

∙ Transcriptional activation complex (KEY FEATURES) o Binding of transcriptional activators to distant enhancers o Looping of DNA  

o Association with basal transcription factors  

o Recruitment of RNA polymerase  

∙ Chromatin remodeling complexes prepare the DNA for  transcription by “loosening it up” 

o Chromatin remodeling complexes (CRC) make hidden  binding sites (like enhancers and promoters) accessible to  transcription factors

o CRCs work by repositioning nucleosomes, or by chemically  modifying the histones so that DNA is unwound a little  

o Chromatin remodeling complexes prepare the DNA for  transcription by repositioning the nucleosomes 

o RNA polymerase can now access the DNA and transcribe  the gene  

∙ Gene expression can be affected by chemical modification of the  DNA 

o DNA methylation turns off genes because methyl groups  “disguise” the enhancers and promoters, preventing  transcription factor binding and activation of transcription  

o DNA methylase adds the methyl (CH3) group to cytosine  o Usually CG rich regions are targeted  

o The inactive X chromosome is extensively methylated

 Methylation is a good way of turning genes off (AKA  epigenetic silencing)

∙ Genomic Imprinting  

o Genomic imprinting is an epigenetic phenomenon by which certain genes can be expressed in a parent of origin  specific manner

o The expression differences of the imprinted genes are due  to different methylation patterns; mothers methylate these genes differently than fathers  

o Once imprinted (methylated), the gene remains  

transcriptionally “off” for all of embryogenesis 

o The methylation imprints are erased early in germ line  development in the offspring, and then reestablished  according to sex-specific patterns 

o Embryonic development expects certain genes to be  expressed from father’s chromosomes, and certain ones to  be expressed from the mother’s chromosomes – this is  what imprinting controls 

o The embryo’s DNA is methylated in the colored patches,  meaning genes of these copies of the homologous  

chromosomes are not expressed  

 The embryo effectively has only one functional copy  of those genes  

∙ Prader-Willi Syndrome and Angelman Syndrome  

o Both disorders are the result of a deletion of several genes  on chromosome 15 

o Heterozygotes are affected  

o Prader-Willi Syndrome 

 If the deletion is inherited from the father, Prader Willis syndrome results

 Prader-Willis involves almond-shaped eyes, hunger,  obesity, decreased muscle tone, decreased mental  capacity, and sex glands that produce little or no  

hormones  

o Angelman Syndrome 

 If the deletion is inherited from the mother,  

Angelman syndrome results

 Angelman is neurological; characterized by  

intellectual and developmental delays, sleep

disturbance, seizures, and jerky/flapping hand  

movements, and lots of happy laughing  

o Different methylation (imprinting) patterns on the  chromosome inherited from mom and the chromosome  inherited from dad are the reason for the two very different syndromes  

∙ RNA stability

o mRNAs vary greatly in stability 

 The balance between mRNA degradation and  

transcription of new messages determines the level  of individual mRNAs in cells  

o 5’ caps and 3’ tails contribute to mRNA stability 

o Sequence elements present in some mRNAs cause them to be less stable  

 Quickly degraded mRNAs usually have A-U rich  

sequences in the 3’ UTR

o At least two distinct mechanisms for RNA degradation  appear to exist  

 One of these mechanisms (nonsense-mediated  

mRNA decay) target incorrect mRNAs that have  

premature stop codons  

o The poly A tail and the 5’ cap help stabilize mRNA (in  eukaryotes)

 Decapped or de-tailed RNA is degraded by nucleases ∙ Iron-dependent regulation of translation of ferritin mRNA  o Feritin is the major iron storage protein in cells  

o If iron is low, ferritin is not needed 

 Iron response element binding protein (IRE-BP) blocks translation of the ferritin mRNA

 As iron increases, IRE-BP binds to the iron instead of  to the mRNA  

 When IRE-BP “lets go” of the mRNA, ferritin protein is produced  

∙ RNAs have many functions in addition to encoding proteins  o RNA molecules have many functions in cells:

 mRNA – messenger RNA

 tRNA – transfer RNA – translation

 rRNA – ribosomal RNA – ribosome  

 snRNPs – splicing

 ribozymes – RNA enzymes  

 miRNAs and RNAi – regulate gene expression  

∙ RNAi

o RNAi (RNA interference) is a method by which introduction  of a dsRNA triggers destruction of any matching mRNAs by  nucleases  

o Laboratory Method for RNAi

 RNAi can be used to “knock down” the expression of  almost any gene 

 RNAi is widely used in variety of cells and organisms  o RNAi machinery can detect:

 Viruses with dsRNA genomes

 Viral RNAs that form hairpins

 Viral RNAs that form dsRNA because of bi-directional  transcriptional  

o RNAi in plants is called post transcriptional gene silencing  (PTGS) 

o RNAi-based therapeutics 

 The production of abnormal proteins is the cause of  most human disease

 Today’s drugs act by blocking the action of disease causing proteins

 RNAi creates the opportunity to silence the  

production of disease-causing proteins and therefore, represents a whole new approach for innovative  

medicines for certain diseases  

∙ Genomes include MANY genes that naturally produce small RNAs o These small RNAs are called microRNAs (or miRNAs) o Lots of miRNAs have been found in diverse organisms  o miRNAs might regulate most genes 

o miRNA genes are needed for normal development o miRNAs are conserved throughout evolution 

o miRNAs usually regulate gene expression by blocking  translation 

∙ Monitoring gene expression with beta-galactosidease staining  

∙ Major Concepts 

o Bacterial genes are often grouped together in operons

o Transcription of genes used to metabolize lactose is  controlled by lactose

 Lactose binds and removes the lac repressor allowing transcription

o Transcription is linked to need by various methods of  sensing the presence or absence of a molecule  

 Proteins such as aporepressors, or RNAs such as  tRNAs or riboswitches sense the levels of the  

molecule in the cell  

o Attenuation is a method of controlling transcription through formation of RNA structures – the terminator and the anti terminator

 Position of the ribosome determines whether the  terminator or the anti-terminator forms  

o Eukaryotic genes are regulated at multiple levels, including modification of DNA or chromatin, transcription, and RNA  splicing  

o Transcriptional activators bind to enhancers and attract  RNA polymerase and its associated factors  

o The transcription complex including RNA polymerase, TBP  and TAFs is very large

o Chromatin remodeling complexes allow access of these  factors to genes  

o DNA methylation modifies the chemistry of DNA and keeps  genes turned off  

o The point of all of this is to allow multicellular organisms to  have different types of cells and to respond to different and changing conditions  

o RNAi uses double stranded RNA (dsRNA) to target and  destroy matching mRNA, which prevents translation of the  message

o RNAi is a valuable experimental tool for investinging gene  function in many organisms  

 Scientists can now more quickly assess gene function o The RNAi response of cells to dsRNA is probably related to  the way cells attack RNA viruses using RNAi

o MicroRNAs (miRNAs) are genes encoded by the genome  and are very important gene regulators  

 miRNAs can block translation or lead to RNA  

degradation  

Chapter 10  

∙ Overview of the recombinant DNA technique “cloning”

∙ Recombinant DNA – the joining together of two or more  deoxyribonucleic acid molecules, often from different organisms o The resulting molecule is then usually incorporated into a  living cell, where it replicates  

o Uses  

 Produce drugs/pharmaceuticals 

∙ Insulin, growth hormone, blood clotting factor  

VIII

 Vaccines 

∙ HepB vaccine

 Making genetically modified organisms 

∙ For science: expressing transgenes and  

knocking things out with RNAi

∙ For agriculture

∙ For drug testing

 Synthetic biology 

∙ Making cells do stuff

∙ Making new organisms  

∙ Type 1 Diabetes

o In response to high levels of glucose in the blood, the  insulin-producing cells in the pancreas secrete the  

hormone insulin 

o Type 1 diabetes occurs when these cells are destroyed by  the body’s own immune system  

o Insulin

 Insulin is a small protein consisting of fifty-one amino acids, synthesized and stored within the pancreas

 The protein consists of two chains, A and B

o Producing insulin for diabetes treatment

 Originally, insulin was extracted from animal  

pancreas tissue to treat diabetics

 Now, insulin is produced in bacteria using  

recombinant DNA  

 Get bacteria to express it for you using recombinant  DNA  

o The gene that encodes human insulin has three introns and two exons  

 The insulin mRNA is spliced, and just contains exons   Reverse transcriptase is an enzyme that copies  

mRNA into DNA  

∙ The copied DNA is called cDNA 

o How to get the gene you want from cDNA 

 1. Pick a tissue that expresses the gene you are  

interested in  

 2. Extract mRNA from that tissue  

 3. Copy the mRNA into cDNA using reverse  

transcriptase (in a test tube)

 4. Use PCR to amplify the specific gene (insulin) you  are interested in from the cDNA  

 5. Run gel electrophoresis to purify your cDNA  

sample  

∙ Use PCR to amplify the insulin coding region from a human cDNA  sample 

o Primers designed to the 5’ and 3’ ends of the insulin cDNA  o Run DNA gel electrophoresis to see if your PCR reaction  worked  

∙ PCR amplification of the insulin gene 

o Now, we will put the insulin cDNA into a plasmid so we can  express it in bacteria  

o Carefully cut out the piece of gel that has your DNA in it  o Melt the agarose, and collect the DNA  

 Usually, small spin columns are used to isolate  

columns are used to isolate and purify the DNA  

∙ A plasmid for expressing genes in bacteria

∙ Restriction enzymes cut DNA at specific sequences o The restriction enzyme BamHI makes a staggered cut that  leaves “sticky ends”  

∙ Cloning an insert into a plasmid

∙ How do restriction sites end up at the 5’ and 3’ end of the insert o 1. Restriction sites might naturally exist in the DNA  o 2. Usually the PCR primers are modified to contain  restriction sites  

 Then, when the gene is amplified by PCR, the  

restriction sites are added  

∙ How are these restriction sites used to join DNA fragments? o The sticky ends are able to base-pair, transiently stabilizing the two ends in close proximity

o The enzyme ligase then covalently bonds the DNA  backbones  

∙ DNA ligase is an enzyme that connects the 3’ hydroxyl of the last nucleotide in the plasmid to the 5’ phosphate on the insert to  covalently attach the insert into the plasmid  

∙ Compatible DNA ends can result in circularization

∙ Transformation of bacteria in the lab 

o The plasmid contains an antibiotic resistance gene so that  cells that have taken up the plasmid can be selected  

∙ Screening for plasmids that have an insert using blue-white  screening 

∙ If LacZ is transcribed and translated into a functional B-gal  protein, the cells will turn blue in the presence of X-gal

∙ Screening for plasmids that have an insert using blue-white  screening  

∙ Now, you have picked a white colony; Since PCR can introduce  errors, you want to make sure the insert has the correct DNA  sequence  

∙ Now we have a plasmid that will let us express insulin in bacteria o Purify the insulin – separate it from bacterial proteins  o Test the purity by HPLC and/or peptide sequencing  o Since the mid 1990s “analog” insulin has been used that  has a few amino acid differences from real insulin  

 Analog insulin clumps less and disperses more  

readily into the blood, allowing the insulin to start  

working in the body minutes after an injection

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