Basis of Genetics: DNA, RNA, Chromosomes, and Genes

Griffith

transformation experiment performed on rats and 2 strains of bacteria that cause pneumonia: type R and type S

R is non-encapsulated, avirulent, relatively harmless

S is encapsulated, virulent, severe pneumonia

hypothesized that the transforming agent was an S protein

showed genetic transfer between cells

Avery

transformation experiment, determined that the DNA from type S bacteria was the genetic material responsible for Griffith’s results, not RNA

treated samples with either RNase or DNase to do so, no S transformants produced when treated with DNase

showed genetic transfer between cells and that DNA is the transforming agent

bacteriophage

virus that attacks bacteria and replicates by invading a living cell and using the cell’s molecular machinery

virulent ones composed of DNA and a protein shell

Hershey and Chase

blender experiment, set up two replicates- labelled DNA with 32P and protein with 35S

infected E coli bacteria with two types of labeled T2 bacteriophages

blender allowed for separation of the phage coats from the bacteria

bacteria were lysed to release phage progeny, progeny of phages that were originally labelled with 32P remained labelled, while the progeny of the phages originally labelled with 35S were unlabeled

showed DNA is genetic material

Gierer and Schramm/Fraenkel-Conrat and Singer

tobacco mosaic virus experiment

demonstrate RNA is the genetic material of TMV, used 2 viral strains

showed RNA not protein is genetic material of some viruses

phosphodiester bond

covalent bond between the phosphate group 5’ of one nucleotide and 3’ carbon of the sugar of another nucleotide

very strong, for this reason DNA is remarkably stable

Chargaff

base composition studies

indicated double stranded DNA consists of ~50% purines and ~50% pyrimidines

amount of A=amount of T, amount of C=amount of G

%GC content varies from organism to organism and determines thermostability

Rosalind Franklin and Maurice Wilkins

x-ray diffraction studies, showed DNA is helical structure with distinctive regularities, 0.34 nm and 3.4 nm

how many bonds are between A and T?

2

how many bonds are between G and C?

3

how far apart are base pairs?

0.34 nm

one complete turn of the helix requires

3.4 nm (10 bases/turn)

sugar phosphate backbones are not equally spaced, resulting in

major and minor grooves

DNA replication

in cell nucleus

DNA unwinds and separates into two strands, each becoming a template for making a new strand

proper base pairs are assembled on the template by DNA polymerase

nucleotides connected by DNA ligase to make new strand identical to old one

new DNA has one strand from original double helix and one new strand

RNA

single stranded and shorter than DNA, less stable than DNA

ribose sugar, uracil replaces thymine

structural aspects of cell biology

package dna in an orderly way in the cell nucleus

total length of DNA in human cell is about 2m, but must fit in nucleus with diameter of 5-10 micrometers

physiological aspect of cell biology

DNA is the same in all somatic cells of an organism but 25000 genes make about 140000 proteins

generate many different cell types

organize different cells into different tissues/organs and express different proteins

the genetic code

a nonoverlapping triplet code

key differences in eukaryotic gene expression

three RNA polymerases

monocistronic gene structure (mRNAs encode single gene product)

RNA processing (5’ cap, polyadenylated 3’ end, splicing)

split gene structure (genes contain introns)

prokaryotic genomic organization

genophore, or a chromosome without chromatin

chromatin in eukaryotes

dynamic, not just a scaffold, providing a physiological template of the genome

a genome indexing platform in a way

to relieve repression by this, much more must happen at these promoters

key to process is controlling access to promoters

organization of DNA in the cell nucleus

tightly bound to small basic proteins (histones) that package DNA in an orderly way in the cell nucleus

net result- each DNA molecule has been packaged into a mitotic chromosome that is 100,000-fold shorter than its extended length

packing of DNA into chromosomes

1. winding of DNA around histones to create a nucleosome structure

2. nucleosomes connected by strand of linker DNA like beads on a string

3. packaging of nucleosomes into 30-nm chromatin fiber

4. formation of looped domains

enzyme digestion reveals that

146 bp of DNA is wrapped around histone core complex in all cell types

chromatin condensation changes during cell cycle

during interphase, it is in its least condensed state and appears loosely distributed throughout nucleus

condensation begins during prophase and chromosomes become visible, remain condensed throughout various stages of mitosis

condensation/folding and decondensation/unfolding of this changes during cell cycle through interactions with various protein factors (condensin and cohesin) and mitotic spindle microtubules

condensation process cooperates with the assembly (biorientation) of sister chromatid pairs to make a normal (faithful) segregation of chromosomes

euchromatin

diffuse or loose interphase form of chromosome

stains light, transcriptionally active

uncompacted and active

heterochromatin

condensed form throughout interphase of chromosome

stains dark, transcriptionally inactive

compacted, untranscribed

two types: constitutive and facultative

constitutive heterochromatin

chromatin that is always heterochromatic in all cells at all times (telomeres, pericentromeres)

faculative heterochromatin

chromatin that does not always need to be heterochromatic but can convert to euchromatin when needed

example- X chromosome in female mammals during: during meiosis inactivated X chromosome reverts back to active euchromatin state, otherwise half of the daughter cells would get inactivated X chromosome

centromeric DNA (CEN)

centre of chromosome, specialized sequences function with the microtubules and spindle apparatus during mitosis/meiosis

telomeric DNA

at extreme ends of chromosome, maintain stability, and consist of tandem repeats

play a role in DNA replication and stability of DNA

unique-sequence DNA

repeated DNA, often referred to as single-copy and usually code for genes

repetitive sequence DNA

repeated DNA, may be interspersed or clustered and vary in size

SINEs- short interspersed repeated sequences

LINEs- long interspersed repeated sequences

microsatellites- short tandem repeats

constitute ~45% of the mammalian genome