hybrid discipline combining genetics and biochemistry

gene structure and function at the molecular level

cannot be considered molecular

nature of genes was unkn

transmission genetics - traits from parent to offspring

DNA/RNA structure and function

DNA replication

Transcription and translation mechanisms

gene structure/function/control of expression

gene cloning, genomics, proteomics, and bioinformatics

concluded that inheritance was "particulate" i.e. each parent contributed particles (genetic units, genes) to the offspring 

Medel's work was rediscovered around 1900s

Mendel had predicted that gametes (sex cells) would contain only one allele of each gene instead of two

Morgan proved that chromosomes carry the genes

and that chromosome number should be reduced by half in the gametes 

demonstrated that eye-color in fruit flies was sex-linked

demonstrated two other phenotypes that were sex-linked

Morgan assumed that genes are arranged in a linear manner 

knew that crossing over could occur during meiosis 

the result is an exchange of genes between two homologous chromosomes

the further apart the two genes are on a chromosome, the more likely they are to recombine

predicted that a mathematical relationship exists between distance separating two genes on a chromosome and the frequency of recombination between those two genes

DNA 

Protein 

a nucleotide is made of a sugar, phosphate group and a nitrogenous base

polymers are formed by linking the sugars together through their phosphate groups

transformation experiments similar to experiment preformed by Griffith

demonstrated that DNA was the genetic material of inheritance

S (smooth) strain forms capsules of cells, virulent, kill mouse

R (rough) does not form capsules, avirulent, does not kill mouse

took S, heat killed, mouse lived

heat killed S, mixed with R, mouse died

found live S and R strains isolated from mouse

demonstrated that transformation had occurred

heat killed virulent cells were found to transform avirulent cells to virulent cells

transformed cells could pass the acquired virulence to their descendants as a heritable trait

-          Removed protein from test extract and extract still retained transforming ability

-          Digested test extract with various enzymes

o   Trypsin – digests proteins , mouse died

o   Chymotrypsin – digests different places of the protein, mouse died

o   RNase – cleaves RNA bonds, mouse died

o   DNase – cleaves DNA bonds, mouse lived!

physical-chemical analysis of the purified transforming substance

ultracenterifugation - MW matched DNA

electrophoresis - high mobility matched DNA

UV absorption - match DNA at 260nm

Chemical analysis - N to P ratio of 1.67

DNA was a monotonous repeat of 4 nucleotide base pairs 

questions regarding whether bacterial genes were like the genes of higher organisms

bases are not found in equal proportions

base composition varied between species

found that there are approx the same amount of C-G and A-T

extended Avery et. al

purified transforming materials so that they contained only 0.02% protein 

showed that genetic traits other than R and S could be transferred 

used T2 bacteriophage

phage composed of only DNA and protein 

page DNA was radiolabeled with ³²P (lot of P in DNA)

phage protein was radiolabeled with ³⁵S (lot of S in protein)

attached phage to host cells, removed phage coat by blending

cells contained little S but lots P (separate trials)

Purines - Adenine, Guanine

Pyramidines - Cytosine, Thymine

RNA has Uracil instead of Thymine

Nucleoside and Deoxynucleotside

Adenine

RNA - Adenosine

DNA - Deoxyadenosine

RNA - Guanosine

DNA - Deoxyguanosine

RNA - Cytidine

DNA - Deoxycytidine

RNA - Uridine

DNA - Thymidine

5' end has the phosphate group

3' end has the OH or the next strand of DNA

antiparallel strands - polarity is reversed between the two

Watson and Crick - slytherpuff

Franklin - discovered double helical structure

Chargaff - base pairs C-G and T-A

Linus Pauling - thought DNA was triple helix, but worked on alpha helix of protein 

double helix structure

length of 1 helical turn

how many N bases were present per turn

width of helix

draw structure of G-C pair

draw structure of A-T pair

C-G has 3 hydrogen bonds that form between the nitrogenous bases

A-T has 2 hydrogen bonds

overall patter of the x-ray showed a helix

major groove - big dip

minor groove - little dip

bases are 3.4A apart from each other

a turn is 34A long

width is 20A

Watson and Crick represented B form

form that the sodium salt of DNA assumes under conditions of high humidity (92%)

considered the common form

bps are perpendicular to the helical axis

10 bp per helical turn

has a pitch of 34A

when the humidity is reduced to 75%

bps tilted about 20 degrees from horizontal

11bp per helical turn

pitch is about 28A

DNA-RNA hybrids assumes the A form

when strands of alternating purines and pyrimidines exist as left-handed helicies

backbone appears to zig-zag 

has pitch of about 46A

12 residues per helical turn

and an inclination of 9 degrees

occurs by heating, the presence of organic solvents, high pH, lowering salt content

disrupt the hydrogen bonds

lowering the [salt] that shield the negative charges on the DNA strands from each other, puts strain on the H bonds

can denature at relatively low temperatures

also called DNA melting 

when the two strands separate, the quenching (suppressing) is reduced and the absorbance at 260nm goes from 30 to 40%

plateau off around 90 degrees

significant effect on DNA stability, Tm and density

Tm and DNA density increase linearly

naturally occurring %s range from 22% to 73%

effect on Tm is due to the 3rd H-bond that exists between G-C

reuniting of DNA strands

3 major factors that contribute - temperature, DNA concentration, time

generally Tm -25degree

low enough that it doesn't promote melting but high enough to allow rapid diffusional movement of DNA and waken transient binding between sequence mismatches

rapid cooling of denatured DNA prevents renaturation and is called quenching 

generally speaking, the higher the [DNA] the faster the renaturation

C₀t and C₀t curves

C₀ is [DNA] expressed in moles of nucleotides per L

t is time expressed in seconds

C₀t is the product of concentration by time

C₀t expressed as C₀t_1/2 or "half-C₀t"

represents when they are half annealed

the higher the C₀t value, the higher the complexity

DNA size ranges from a few kilobases to hundreds of megabases

a convenient means to estimate the size of small DNAs is by TEM

TEM also shows if the DNA is linear, circular, or supercoiled

DNA size is more commonly estimated by exectrophoresis

C-value = DNA content per haploid cell

you would expect that the more DNA an organism has in its cells the more complex the organism and the more genes it has

C-value paradox is generally explained not by extra genes, but by extra noncoding DNA

a reliable way to estimate the number of genes in an organism is to estimate the number of different mRNAs

Adenosine 5'-monophospate

Guanosine 5'-monophosphate

two parent purine nucleotides of nucleic acids

de novo purine nucleotides biosynthesis starts with the molecule phosphoribosyl pyrophosphate (PRPP)

starts the pathway from scratch

1st step - an amino group is donated by the glutamine and this group attached to the C-1

the resulting 5-phosphoribosylamine is extremely unstable

(half-life of 30s at pH 7.5)

the remainder of the purine ring is built upon this structure

IMP

the first intermediate in the de novo purine pathway to have a complete purine ring

AMP and GMP are then made from IMP

are synthesized from aspartate, PRPP, and carbamoyl phosphate

cytidine 5'-monophosphate (CMP) and uridine 5'-monophosphate (UMP) are made in this pathway

the pyrimidine ring is made first then it is attached to ribose 5-phosphate

purines are degraded  yields uric acid

pyrimidines are degraded yields urea

free purine and pyrimidine bases formed from the degradation of nucleotides, salvaged, and reused to form "new" nucleotides