Week 3 Flashcards
types of rna
mRNA—code for proteins
rRNA—Core of ribosome
in vitro
in glass, in test tube
in vivo
in life, without disrupting organism
Nirenberg experiments
make a cell free extract from E. coli
added different parts at different times (similar to electrophoresis)
use 14C as a tracer to label proteins (allows to track very small amounts)
show protein synthesis in vitro (add amount of soluble RNA, show that more proteins occur, add ribosomal rna—necessary)
mRNA—needed for protein production in a dose dependent manner
mRNA—needed for protein synthesis once endogenous rna removed by DNAse (can stimulate, once DNA destroyed)
x-ase
short way to say an enzyme that destroys x
penicillinase—enzyme that destroys penicillin, etc.
DNase—enzyme that destroys dna, etc.
how did scientists find genetic code?
idea: take simple synthetic rna (UUU poly-U), then see what this codes for
poly U results in poly phenylalanine peptide
very simplified system
make poly C, poly A, etc, in each case only single amino acid made
can’t make poly G, since makes secondary and tertiary structures—not really translatable
UC… gives 2 amino acids, so triplicate code, etc.
UA… gives 4 amino acids
Khorana
did experiments on genetic code, chemically synthesized nucleotides
wobble base
third base in nucleotide sequence, changes
UC together, AG together for wobbling
directionality of mRNA synthesis
5’ terminus of mRNA first, then 3’ terminus last
during protein synthesis, same direction
but reading of dna is 3’ to 5’
correspondence between tRNA and codon
not a clean (one to one) correspondence, always
UAA, UAG, UGA
stop codons—mean stop, terminator, nonsense
if mutation changes base of codon
1) no change—e.g wobble base, synonymous mutation
2) change one amino acid to another—nonsynonymous mutation
3) changes amino acid to stop—nonsense mutation, or stop gain
dna structure
dna is wrapped around histones in large coils in the cell
- special enzymes needed to uncool and recoil dna
- dna is very long molecule that is highly condensed
chromosome
highly packed structure of dna
each species has a characteristic number of chromosomes (46 for humans)
-22 autosome pairs, 2 sex chromosomes x and y
when cell divides, chromosomes are visible
nondividing cell—tangled mess
two parts—chromatid and centromere
chromatin
when eukaryotic cell is not dividing, dna is tangled mass of thin threads
material from which chromosomes of eukaryotes are composed
consists of protein, rna, dna
the cell cycle
organization of how dna and life of cell is organized
most of cell happens during interphase (dna replication as chromosomes duplicate, protein production, etc.)
then growth phase, and prepares to divide (mitosis, cytokinesis—movement into 2 cells)
orderly sequence of events that occurs in time
stages: G1 (cell increases in size), S (copies it’s dna), G2 (prepares to divide), and M (divides)
- interphase —G1, S, G2
mitotic phase
where cell division occurs
four phases: prophase, metaphase, anaphase, telephase
interphase
majority of time for cell
late interphase—when chromosomes begin to compact
end of interphase—two copies, so four copies of each gene (one on each chromosome)
G1, S, G2
no observable changes under the microscope
early and late prophase
early prophase—nuclear membrane becomes in distinct, chromatin fibers more packed and condensed
late prophase—nuclear membrane and nucleosus vanish completely
metaphase
short moment during which chromosomes look like we see (in well known shape)
chromosomes become attached to spindle fibers
then anaphase, then telophase
karyotype
number of chromosomes
species specific
chromosomes can be very small of very late, different shapes
species differing by chromosome number cannot interbreed
diploid number
number of chromosomes found in somatic (non sex) cells
haploid number
one chromosome of each kind
autosome
non-sex chromosome
22 autosome pairs (not sex chromosomes)
receive one of each autosome from father, one of each from mother, X chromosome from m, etc.
meiosis
reduced the chromosome number such that each daughter cell (egg or Sperm) has only one of each kind of chromosome
ensures that the next generation…
two nuclear divisions, result will be four haploid nuclei (each is different but complementary)
germ cell (sperm/egg) combine to form zygote, then make somatic cells and germ line cells
M1 and p1 side by side, etc.
increases diversity in offspring
chiasma
maternal and paternal chromosomes exchange material
point at which paired chromosomes remain in contact during first metaphase of meiosis
genetic recombination during meiosis
1) crossing over—non sister chromatids exchange material
- happens on average once or twice on each chromosome arm
2) independent assortment—homologous chromosomes distributed to daughter cells randomly
meiosis vs mitosis
before each, dna separation occurs only once during interphase
Mitosis requires one division
Meiosis 2 divisions
2 diploid daughter cells from mitosis
-generically identical to parent cells
4 haploid daughter cells from meiosis
-not genetically identical to parental cells
…
human life cycle requires both
meiosis—spermatogenesis in males
—oogenesis in females
mitosis involved in growth of child and repair of tissues
zygote
cell made from 2 germ cells
46 chromosomes
23 pairs of homologous chromosomes
dna replication
semi conservative
- each new dna molecule consists of half of the old, half of the new dna
every time, split in half and one half in one cell, other half in another cell
since dna strands antiparallel, only one strand will form a continuous copy
dna synthesis
always occurs 5’ to 3’
DNA polymerase involved in replication
requires energy—phosphate
lagging strand of dna
forms a series of short pieces with gaps
During synthesis
Okazaki fragments, require use of other enzymes to complete process
Okazaki fragments
ligation ties several Okazaki fragments together
other strand just polymerase (continuous)
origins of dna replication
where dna replication is originated
bubbles—open up in both directions
replication forks
Okazaki fragments in bubbles
polymerases
DNA polymerase requires a primer to start polymerization reaction
primer—dna primase makes primer, made out of rna
dna helicase
unwinds dna, enzyme
telomerase
adds additional repeats to template strand
helps with end replication problem on lagging strand
end replication problem
telomerase solves this
using rna template, elongates parent strand and makes lagging strand longer
error rates of DNA polymerase
very low, but large genome so
proofreading: several mechanisms eliminate most of these
end error rate 1/10^9 (only a few mistakes)
pcr purpose
isolate and amplify a specific piece of dna from a given mixture
-eg gene in full genome (circ. Rhythm), gene in mix of bacteria, presence of viral dna)
identify presence of dna in a mix
introduce subtle changes into piece of dna
amplify all dna from small amount
sequence piece of dna
amplify selectively one pice if dna for genotyping, etc.
pcr history
1983: recombinant dna technology existed
realized principle of dna replication
-> can be used to target specific DNA sequence for exponential amplification and further analysis
pcr process, chain reaction
double stranded dna
heat to separate strands
hybridization of primers (primers on each strand, going in opposite directions)
add dna polymerases, etc
dna synthesis from primers
then do same on each new strand—second cycle. Then continue—exponential
needed for pcr reaction
dna starting material
primer set (2 primers)—min length of 16
Dntps
Enzyme
Buffer, salt
