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Molecular Biology

- bacteria and eukaryotes are composed of double-stranded DNA

- some viruses are composed of single or double stranded RNA

Historical Timeline

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- Meischer discovery that DNA in nucleus

- Gates UV of 60nm kills bacteria

- Avery and MacLeod transformation of rough streptococcus to smooth using purification of nucleic acid and RNase digestion

- Lederberg accurate genetic map of E. coli based on conjugal mating

- McClintock transposable elements = jumping genes

- Chargaff hydrolyzed DNA and separated bases by paper chromatography to show base composition varied with different DNA sources

- Franklin used x-ray crystallography to discover sugar-phosphate backbone of DNA and elucidated helical structure

- Hershey and Chase differentially labeled T4 phage

o Labelled protein with sulfur

o Labelled DNA with phosphorous

o Phosphorus detected in cell

- Watson and Crick DNA had two helical strands, hydrogen bonded together

- Ability of E.coli to mate is by F factor which is an infectious particle not associated with the chromosome

- Jacob and Wollman propose Episome to describe extrachromosomal element but now called plasmid eg. F, colicine, phage lambda

- DNA cast with heavy metals to enhance visualization because so thin

- Multiple drug resistance transferred by an episome designated the R-factor

Plastid and Mitochondrial DNA

- Plastid � plant storage and photosynthetic organelles

- small double-stranded DNA present at several copies

- Mitochondrial DNA is linear in plants and fungi but circular in humans and inherited from mother

- Chloroplast DNA is circular

- Protein synthesis is inhibited by prokaryotic inhibitors eg. Chloramphenicol


- usually double-stranded circular extrachromosomal DNA elements found mostly in bacteria

- sometimes able to find single-stranded replicative intermediates in cell indicating rolling-circle mode of replication

- nonessential

o can be eliminated by ethidium bromide (intercalating dye) or acridine orange

§ differentially affect replication of plasmid

- vary in copy number

- smaller than chromosome

o carry function for replication

- two possessing similar replicons cannot exist in same cell because incompatible

- Plasmid Associtated Genes

1. resistance genes

. virulence factors � toxins

. metabolic pathways � ability to metabolize certain carbonaceous compounds such as toluene

4. symbiotic � ie. Nitrogen- fixing bacteria on legumes

5. conjugal transfer systems � transfer genes on plasmid F encode pilus synthesis and mediate conjugation between cells

NOTE pBR, pUC , pGEM are nonconjugative plasmids

6. Restriction and modification systems � EcoRI

- Cairns labeled E.coli DNA by growing cells with H-Thymine and lysing the cells to trap DNA and exposed to silver halide emulsion

o Observed circular tracks which led to belief that prokaryotic genome was single circular chromosome

- rate of reassociation of fragmented denatured DNA is a function of complexity Cot Analysis

- Berg first cloning reaction

- Sanger first sequencing technique using dideoxynucleotides to sequence SV40

- Why Sequence Genomes?

o Identification of basis for disease states

o Biotechnology

o Categorization and taxonomy

o Pure science

§ No more than 0-5% of organism’s genes can be identified by classical genetic techniques

Genetic Mapping Techniques

A. Top-Down approach

- For more complex prokaryotic DNA

- cutting genomic DNA with restriction endonucleases and separating fragments by standard agarose gel electrophoresis is more easily done for plasmids and viral DNA, but not for the more complex DNA

- joining fragments

o Linking clones rare clones carrying restriction site (ie. of I-CeuI)

o Two-dimensional gel electrophoresis

§ Every site in DNA molecule is clipped

§ DNA partially and completely digested

0. Undigested run as circular piece

- Extract linear DNA and religate clone to get overlapping DNA on each side of rare endonuclease site

o Use to make library of genome

- Three developments in physical mapping

o in situ lysis techniques

§ minimize shearing

§ cells embedded in agarose blocks and lysed with enzymes (ie. Lysozyme) with mild detergents

§ proteinase K digestion removes proteins à intact DNA

o discovery of restriction endonucleases that recognize rare sites rare cutters

§ I-CeuI is an intron-encoded endonuclease in chloroplast rRNA gene

o Development of alternative gel technologies

§ The standard agarose gel electrophoresis will separate molecules up to a size of 50kb, but any larger molecules run at same mobility due to raptation (end-on migration through gel)

§ Pulsed Field Gel Electrophoresis � periodically alters orientation of molecules to avoid raptation

1. Doesn’t separate circular molecules

- Choice of enzyme is affected by

o genome size

o presence of methylated bases which can affect certain endonucleases

o GC content

§ NotI and AseI not useful for high GC

§ SwaI, PacI and PmeI not good for low GC

- Advantages

o Simple

o Accurate picture of genome

o Can be coupled with Southern Hybridization

§ For prokaryotic cells to establish genetic maps

B. Bottom-Up approach

- for very large genomes of higher eukaryotic organisms

- using vector systems

- the size of the library is a function of the mass of the genome, the insert size and the probability that all genes are represented in the library

- Genomic DNA cloning

o Isolated DNA from organism fragmented by partial digestion with restriction endonuclease

o Molecules separated by size and ligated to vector

o Transformed into intermediate host cell

- Vector Systems

o Cosmid Cloning Vectors

§ SuperCos

§ Based on cos region of bacteriophage lambda DNA which is involved in the in vitro packaging of phage DNA into phage particles

§ Terminase cuts enzyme at this site and packages the DNA into the phage head

§ Size of DNA insert is critical

. Cannot exceed capacity of phage head = 51kb

§ Phage particles more efficient transfer of DNA to cells than transformation

§ Method

. Supercos cut with Xbal to linearize vector

4. BamHI cut to produce left and right arm

5. Add insert and ligate to arms using ligase à Concatemer

6. Concatemers packaged with lambda packaging mix into phage particles

o BAC System

§ Bacterial artificial chromosomes

§ Based on F plasmid of E.coli

§ Can maintain 1Mb of insert stably

§ Inserts up to 50kb

§ No cells carrying two different BAC clones

§ Low frequency chimeras

§ Very stable

o YAC System

§ Yeast artificial chromosomes

§ Shuttle vectors capable of replicating in bacterial and yeast cells

§ Possess ColEI replication origin and an anibiotic resistance marker

§ Origin provided by autonomously replicating sequences (ARS) found in all chromosomes

§ Cloning selection based on nutritional markers not antibiotic resistance

7. Its presence in cells allow for yeast to grow on minimal media if auxotrophic

§ Have a centromere and telomeres

§ Advantage with eukaryotic genomes

§ Cohen used YAC clones to construct first physical map of human genome

§ Problems

8. High frequency of chimeras = two unrelated fragments in same vector

. More difficult to work with yeast than E. coli

o Transformation frequency 1000x lower

o More difficult to isolate

10. High frequency of two different YACs in same cell

11. Inserts frequently unstable

- Characterization of clones done in many ways

o Restrction endonuclease digestion pattern � vector sequences will show up as a constant in all clones

o Riboprobes � labelled probes of each end on the cloned sequence and hybridize these to each clone à contigs = ordered array of overlapping clones (physical map made up of clones)

- capillary gel electrophoresis permits rapid DNA sequencing

- Venter set up NIH

o Sequenced bacterial genome of human pathogen

o Used whole-genome shotgun sequencing approach

§ Randomly shear DNA mechanically using neubulizer (1.5-kb)

§ Fragments are cloned into vector that was cut with enzyme

§ Random shearing leads to random ’ and 5’ extensions which require polishing before cloning which is done with T4 DNA polymerase

1. Possesses polymerizing activity and ’à5’exonuclease activity

§ Vector ligated with clone transformed into E. coli

§ Sequence DNA preparation with two vector specific primers and get sequence from ’ and 5’ ends of cloned fragment

§ Use computer to look for similarities and produce contigs

§ Variety of techniques available for gap closure including PCR

§ Edit and correct mistakes


- what can one do with the sequence data?

o Genomic Mass

o Genomic Shape � linear or circular

o GC% content

§ Regions of higher or lower than average could indicate horizontal gene transfer

o Restriction sites

o Repeats

§ Direct repeats

§ Inverted repeats

§ Mirror repets

§ May represent sites at which proteins bind to

§ Prokaryotes have less redundancy than eukaryotes in sequence

§ Many are restroposons = DNA elements that are mobile through RNA intermediate i.e. Alu sequences

§ Two cases in which sequences encode useful genes for proteins and rRNA’s

1. Histone Gene Family

o Encoded by multiple gene copies lacking introns

o Nontranscribed spacers between histone transcriptional units

o Sequence of gene highly conserved

. rRNA genes

o gene order in prokaryotes is almost always rrs � rrl � rrf ( a single polycistronic transcript is enzymatically processed into the correct sized rRNAs)

o growth rate correlated with copy number for E. coli

o in eukaryotes transcripts are longer and contain less information

o dinucleotide frequence

o Termini can have great variation

§ Terminal redundance � may be direct or inverted and permit circulization or concatmer formation

§ Cohesive ends � certain phages possess 5’ or ’ complementary termini

§ Terminal proteins � associated with adenovirus genomes

§ Snapback structures � closed hairpin end in double strand

Introduction to Genes

- Initiation codons fMet

- Termination codons

o Ochre TAA

o Opal TGA

o Amber TAG

- Shine-Dalgarno region is a subset of TAAGGAGGT found 4-10 nucleotides upstream (5’) to gene

- Homology � implies that compared sequences diverged from common origin

- Paralogs � homologs produced by gene duplication from divergent evolution

- Orthologs � homologs produced by speciation

- Xenologs � homologs resulting from horizontal gene transfer

- Humans have 50-100kb/open reading frame

- With smaller genomes there is a linear relationship between genome size and open reading frame number

- Overlapping genes exist such that the initiation codon of one open reading frame overlaps with the termination codon of the previous one

o One sequence encodes two genes

o Good for conserving space

- cDNA is a copy of mRNA

o obtain information about not only the open reading frame but the upstream and downstream sequence

- split genes are a result of introns separating exons (the coding regions)

o mammalian mitochondrial DNA lacks introns

o lower eukaryotic mitochondrial DNA have introns

§ fewer and shorter



- Minimal genome determined to sustain life

- Comparative genomics

o Single Nucleotide Polymorphisms

§ Humans are .% identical

§ Occur once every 150bp

§ Unevenly distributed

§ Only 000 studied occur in protein encoding or regulatory regions


- Wilkins and Williams coined term “proteome” = the protein complement expressed by a genome or tissue


- developed as a result of PCR amplified genes being able to fix to glass surfaces

- less labor intensive and expensive than Protemoic procedures

- could use to determine which genes are turned on or off under a variety of conditions using flourescently labeled cDNA probes

Chromosomes of Eukaryotes

- localized within nucleus

- dispersed as chromatin

o composed of equal amounts of DNA and protein

o histone plus nonhistone proteins

o gently osmotic lysis releases material for observation under Electron Microscope

§ see “beads on a string”

§ winding of DNA around proteins reduces contour length by about 7x and introduces negative supercoils

§ nucleosome � contains HA, HB, H and H4 and 146 bp of DNA

· DNA wound around core of histone

· 1.75 left-handed superhelical turns around protein octamer

· linker between H1 � H5

o tightens the winding of nucleosome

o increases packing ratio by about 40 - 100

· will join together to form solenoid and then hyperfolds to form rosettes

- DNA helix à histones à nucleosome à solenoid à rosettes

- Minimum # of chromosomes Ant

- Maximum # of chromosomes Fern family

- human genome = 1 metre in length

- gently lysis with low ionic strength detergent

Chromosomes of Prokaryotic Cells

- genomic DNA constitutes for 4% of cellular dry weight

- genomic DNA concentrated within cell in bodies called nucleoids which occupy only a portion of intracellular volume

- Cairns’ DNA = large covalently closed, circular, naked DNA

o RNA polymerase main associated protein

o viscous

- condensed or folded

- internal environment of cell where DNA is located is high in proteins, polyamines (putrescine, spermidine) and ions such as K and Mg (counteract repulsive forces)

o in humans sperm histones are replaced by highly basic protamines

- gently lysis with mild detergent in presence of spermidine (to neutralize DNA results in a nonviscous lysate)

o showed highly folded chromosomes

o mostly protein and DNA

- if nick DNA with Dnase, whole genome doesn’t unwind à thus constrained into independent domains (about 40)

- nucleosome-poor

- viral, mitochondrial and plastid DNAs are histone-free

- dinoflagellate chromatin has low protein content

- contain variety of DNA-binding proteins

o homology of histones major ones in E. coli

§ HU

§ H-NS

o Reduce contour length

o Nonessential

- Zimmerman and Murphy high protein concentration in bacterial cells à macromolecular crowding and genomic condensation

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