genetics 2 Flashcards
transcription occurs in nucleus, mRNA from DNA, antisense strand is used to transcribe RNA from 3’ to 5’
translation occurs in ribosome, protein is made, codons read from 5’ to 3’
central dogma (francis crick)
codons read in unbroken chain, groups of codons called reading frames, clear start and stop location
continuity
each amino acids is associated with tree possible codons, first two bases are what matter, third is wobble position
redundancy
third letter of codon, may code for same amino acid regardless, protects against mutation e.g. point mutation, still codes for same amino acid, contributes to efficiency
wobble position
genetic code is the same in all living organisms bacteria to animals, evolutionary significance, indicates common ancestor
universality
not evenly spaced, inherited, length and number of chromosomes not related
genes
increase or decrease gene expression, e.g. promoter sequences which help to determine which genetic processes will be activated
regulatory regions
binding site for the apparatus that copies a gene and it can either block or enhance binding, determining when a specific gene will be expressed
promoter sequence
exons, code for a particular polypeptide product
coding regions
introns, interspersed with exons
non coding regions
developmental and regulatory functions, regulate gene expression, can silence or initiate gene activity and is correlated with developmental complexity
introns
a gene can code for more than one polypeptide product by copying different combinations of exons
alternative splicing
frequency (more) and length (longer) of introns is positively correlated with the developmental complexity of an organism
developmental complexity
repetitive sequences of DNA that contain base pairs that repeat over and over - minisatellites and microsatellites
variable number tandem repeats (VNTRs)
high mutation rate, leading to diversity in population, e.g. telomeres and centromeres
minisatellites
shorter than minisatellites, show variation in length (# of repeats) among individuals, can be used in DNA fingerprinting, paternity testing, forensics etc.
microsatellites
clusters of genes that are the same/very similar and are located in a specific region, usually code for products in high demand e.g. genes that code for histone proteins
multigene families
jumping genes, sequences of DNA randomly inserted throughout genome, contribute to existence of multigene families: are either LINES (long interspersed nuclear elements) or SINES (short interspersed nuclear elements)
transposons
copies of the genes of multigene families that have mutated to the point where they no longer function
pseudogenes
mRNA, snRNA, tRNA, rRNA
types of RNA involved in protein synthesis
messenger RNA, carries a blueprint of DNA
mRNA
small nuclear RNA, joins with proteins to create spliceosomes which cut out introns and join exons together in mRNA
snRNA
transfer RNA, clover leaf shaped molecule that carries amino acids to the mRNA for polypeptide formation
tRNA
ribosomal RNA, forms the active rivosome and assists in reading the mRNA
rRNA
initiation, elongation, termination
transcription steps
antisense strand selected to synthesize, transcriptions begins at the promoter sequence when transcription factors bind to the TATA box, RNA polymerase begins working, 5’ to 3’
initiation
promoter sequence, rich in T and A, binding site of transcription facors
TATA box
completed assembly of transition factors and RNA polymerase on the promoter
transcription initiation complex
rna polymerase moves along DNA, unwinding a section and synthesizing mRNA from 5’ to 3’, adding nucleotides, double helix reforms after RNA polymerase passes through and the mRNA strand separates from the DNA, more than 1 mRNA can be created at a time due to many RNA polymerases
elongation
RNA polymerase does not need rna primers, does not proofread (transcription less accurate), rna polymerase subs T for U
differences between transcription and DNA replication
termination sequence (rich in G & C followed by string of A on antisense strand) signals for stop, G&C form base pairs with itself and fold into hairpin loop structure, RNA polymerase separates from DNA antisense terminating transcription and RNA polymerase can now bind to another promoter sequence
termination
needs to undergo processing, 5’ end is capped with a 5’ cap of 7 guanine nucleotides and a long series of adenine nucleotides are added to the 3’ end of mRNA (poly A tail) and mRNA splicing occurs to remove introns
precursor mRNA
7 guanine nucleotides, protects it from degenerative enzymes and serves as the initial attachment site of mRNA for translation to occur
5’ cap
protects mRNA from degradative enzymes in cytoplasm, added by poly A polymerase
poly A tail
spliceosome (premRNA, proteins and snRNA) cleave premRNA at ends of each intron, splicing together exons by causing introns to loop out
mRNA splicing
base pairing btwn complementary nucleotides make clover leaf shape, has 2 binding sites one for mRNA and one for amino acid
transfer RNA (tRNA)
anticodon has a nucleotide triplet that is complementary to mRNA codon, anticodon can pair with more than one codon due to third nucleotide in wobble position
mRNA binding site
accepting end, 3’ end, binds to specific amino acid determined by anticodon mRNA pairing
amino acid attachment site
tRNA attached to amino acid
amino-acytl tRNA (aa-tRNA)
site of translation, made up of two subunits large and small
ribosomes
contains 2 linear strands of rRNA and abt 30 proteins
large subunit
1 linear strand of rRNA and abt 20 proteins
small subunit
A site, P site, E site
sites on active ribosome
exit site
E site
polypeptide (peptidyl-tRNA) binding site
P site
amino acid (amino acyl tRNA) binding site
A site
with help from initiation factors (IF), small subunit of ribosome binds to a leader sequence just ahead of the AUG, next initiator RNA with anticodon UAC and methionine base pairs with AUG with help from initiation factors, initiator aa-tRNA, mRNA and small subunit of the ribosome form the initiation complex, large subunit of the ribosome
initiation
located in the p site of the ribosome
initiator aa-tRNA
another aa-tRNA then base pairs with the next codon on the mRNA at the A-site, methionine from the initiator aa-tRNA is covalently linked to the incoming amino acid forming a peptide bond between the amino acids btwn aino acids, ribosomes moves along mRNA from 5’ to 3’ to the next codon translocation, initator tRNA moves into the E site (released) incoming aa-tRNA moves to P site, A site now open for the arrival of the next aa-tRNA
elongation
when ribosome reaches mRNA stop codon in A site, release factor binds in A site, polypeptide is released from tRNA, tRNAs are released from ribosome and will be used again, ribosome separates into its large and small subunits which will be used again
termination
many ribosomes bound to one mRNA
polyribosome (polysome)
always needed and always being transcribed/translated
housekeeping genes
turning specific genes on or off
gene regulation
transcriptional, post transcriptional, translational, post translational
gene control types
regulates which genes are transcribed, controls rate of transcription (introns)
transcriptional
introns are removed and exons are spliced together to create mRNA
post transcriptional
controls rate of translation and lifespan of mRNA strand (poly A tail)
translational
controls the lifespan of the active functional protein
post translational
recognized by regulatory proteins that bind to DNA sequence to control transcription
regulatory sites
e. coli has lac z, which codes for b-galactosidase (splits lactose into glucose and galactose) on the lac operon, which is made of 3 genes (lac z, lac y, lac a) that code for proteins, promoter and regulatory sequence - operator. operator functions as a control, either allowing RNA polymerase to begin transcription or preventing transcription from occurring, inducible operon
operon model (bacteria)
in absence of lactose, repressor protein (lac I) binds to operator region, making it impossible for RNA polymerase to bind to the promoter, no transcription of lac operon gene.
if lactose is present, it binds to lac I repressor, causing the repressor to change shape and detach from the operator site. Lactose acts as an inducer (signal molecule) and RNA polymerase then binds to promoter sequence and transcription can occur
negative gene regulation in lac operon
repressible operon, contains 5 genes, a promoter and an operator sequence. Outside operon, separate gene codes for a repressor protein. Normally e. coli makes enzymes that synthesize tryptophan but if tryp is present, it will bind to repressor (tryp now a corepressor molecule) changing shape and allowing repressor to bind to operator, blocking transcription
co repression in tryp operon
altering a DNA sequence and multiplying a particular gene or gene products
genetic engineering
enzymes found in bacteria that act as molecular scissors and can cut double stranded DNA at its recognition site by disrupting hydrogen bonds between bases and phosphodiester bonds in backbone
restriction endonucleases
PALINDROMIC !! usually 4-8 base pairs long and have 2 fold symmetry (same when read 3’-5’ and 5’-3’)
recognition sites
short single stranded overhangs lacking complementary bases, can be joined tgt with another fragment cut by the same restriction endonucleases through complementary base pairing
sticky ends
restriction endonuclease cleaves btwn nucleotides opposite each other, no overhang
blunt ends
dna made up of different organisms spliced together
recombinant dna
naturally attracted by complementary base pairing and hydrogen bonding, then DNA ligase reforms the phosphodiester bonds through condensation reactions
sticky ends rejoining
dont have natural attractions, must use t4 DNA ligase
blunt ends rejoining
to protect bacteria from viruses by cutting up foreign DNA, needs to recognize own DNA (through methylation)
role of restriction endonucleases
methylases are enzymes that add a methyl group to DNA at recognition site, no longer recognized by restriction endonuclease
methylation
independent of bacterial chromosome, lack a protein coat, small, exist naturally in cytoplasm of bacteria, can be shared btwn bacteria through bacterial conjugation
plasmids
insert foreign gene in plasmid, so that it produces that gene, then restriction endonuclease is used to create sticky ends, inserted into plasmid, recombinant DNA is formed (how insulin is made)
transformation
plasmid that contains foreign DNA
vector
change in DNA of an organism can be harmful - genetic disorder, neutral/silent or benefit
mutations
occur in gonads and may be inherited, most mutations occur in somatic cells and aren’t inherited
genetic mutations
due to errors in DNA replication
spontaneous mutations
from exposure to mutagens directly altering DNA in cells
induced mutations
substance that can cause mutations and directly alters the DNA in cells
mutagen
radiation, uv light, x rays
physical mutagens
tobacco smoke, pollutants e.g. carbon monoxide, base analogs (mimic DNA nucleotides) , intercalating agents
chemical mutagens
one base or small grp of bases, substitution, insertion, deletion, inversion of two adjoining base pairs
point mutations
insertion or deletion of a base
frameshift mutations
translocations - movements of entire genes from one chromosome to another
chromosomal mutations
triplet of codons
reading frame
nucleic acid fragments are separated by their differing lengths
size separation
DNA is negatively charged bc of phosphates and will move toward positive electrode
charge separation
gel box containing agarose gel covered in buffer solution, prevents a medium for flow of electric current and prevents overheating and drying out, placed between two electrodes and the wells of the gel are at the negative electrode
gel electrophoresis
dna fragments of known size placed at loading wells of negative electrode, that can be used for comparison aka DNA ladder, loading dyes are used as well
molecular markers
ethidium bromide, chemical that inserts itself and binds to DNA carcinogenic and fluorescent under UV light
DNA staining
created dna sequencing used to determine sequence of bases in a dna strand and won nobel prize
sanger
chains of DNA sequences are terminated when dideoxynucleotides ddNTP are added, which prevent the binding of the next nucleotide, don’t have a hydroxyl group on 3’ end of sugar and therefore nucleotide cant attach and dye will make fluorescent identifying which base terminated sequence
sanger sequencing
so that entire sequence can be analysed and multiple lengths of the chain will be created
4 diff ddNTPS
restriction enzyme digestion
cutting dna
in vitro polymerase chain reaction
amplifying dna
gel electrophoresis and dye staining
visualizing dna
sanger sequence
sequencing dna
create millions of copies, Dr. kary mullis, uses taq polymerase as it can withstand high temps and not denature unlike dna polymerase iii
polymerase chain reaction
denaturation to break hydrogen bonds + separate strands, primer annealing (2) are added to each strand and anneal to complementary sequences at target, elongation polymerase attaches to primers to create complementary copies of target sequence on both strands
pcr cycle
paternity testing, forensics testing
applications of pcr and dna sequencing
