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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
Write my Essay on Molecular Biology for me
- 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
Plasmids
- 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
Bioinformatics
- 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
POST GENOMICS
Genomics
- 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
Proteomic
- Wilkins and Williams coined term “proteome” = the protein complement expressed by a genome or tissue
Transcriptomics
- 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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