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Chapter 8.6-17.3 plus lab terms

Transformation

genetic recombination with DNA

bacteriophage

a virus that infects bacteria

transduction

genetic recombination after phage infection

lysogeny

virus repressed and incorporated into the bacterial chromosome

prophage

the viral DNA that integrates into the chromosome

temperate

viruses that can either lyse cells or act as a prophage

virulent

viruses that only lyse cells

lysogenic

a bacterium with a prophage

DNA and RNA nucleotides

consist of a sugar, a base, and a phosphate group

x-ray diffraction of DNA

the experimental evidence of DNA structure

Gel electrophoresis

separating DNA fragments by size

Avery, MacLeod, and McCarty's experiment

DNA is the 'transforming principle'; used ribonuclease, DNAase, protease.

Hershey/Chase experiment

DNA directs the reproduction of bacteriophages; labeled protein coat and labeled DNA

Meselson-Stahl experiment

evidence that DNA replication is semiconservative in bacteria; used nitrogen isotopes.

semiconservative replication of DNA

one old, one new

replicon

length of DNA replicated from one origin

gyrase

a DNA topoisomerase that relaxes supercoiling downstream by making cuts that undo twists and knots

RNA primer

synthesized under direction of an RNA polymerase called a primase

Primase

RNA polymerase

Fluorescent in situ hybridization (FISH)

can be used to detect chromosomal translocations

chromosome painting

combination of probes produce a unique color pattern for each pair of homologous chromosomes

single stranded binding proteins

help keep the replication bubble open

DNA synthesis

removing the RNA primers and filling the gaps

telomeres

protect chromosomes from fusion or degradation

Tetrahymena

telomerase was discovered by Blackburn and Greider in _________; a single-celled eukaryote.

telomerase

only found in cells that undergo extensive proliferation (embryonic cells, germ cells, stem cells)

reverse transcription

DNA transcribed from RNA

synapsis

pairing of homologous chromosomes during meiosis

degenerate

most amino acids can be specified by more than one codon

frameshift mutation

an insertion of one nucleotide shifts

Wobble rules

only minimum of 30 different tRNAs needed to specify 61 triplets

promoter

specific DNA sequences upstream of a gene

introns

eukaryotic RNA transcripts contain nucleotide segments that are not found in final mRNA product

Snurps

small nuclear RNAs (snRNAs) complexed with proteins

amino acids

subunits of proteins

radical groups

gives tRNAs their chemical identity

Beadle and Tatum

induced and isolated Neurospora mutants that showed auxotrophic growth

protein secondary structures

arrangement of amino acids within certain sections of protein

quaternary level

for proteins made up of more than one subunit that are associated through non covalent bonds

point mutation

single nucleotide substitution that can be a missense, nonsense, or silent mutation

missense mutation

codon now encodes a different amino acid

nonsense mutation

stop codon inserted instead

silent mutation

same amino acid due to 3rd position wobble

Spontaneous mutations

arise from replication errors or tautomer shifts or depurination and deamination

replication errors

wrong nucleotide inserted, slippage in repeat regions

tautomer shifts

(T, G keto to enol; A, C amino to imino) cause anomalous base-pairing

induced mutagens

arise from chemical mutagens, UV light, ionizing radiation

xeroderma pigmentosum

recessive disorder than predisposes individuals to UV-induced DNA damage and skin cancer; mutations in at least 7 genes involved in nucleotide excision repair can cause this disorder

proofreading

DNA polymerase III (bacteria) can also act as 3' to 5' nuclease

mismatch repair

repair enzyme recognizes mismatch in newly synthesized strand

RecA protein

directs recombinational exchange

Ames test

screens compounds for their mutagenicity in Salmonella bacteria

ethylmethane sulfonate (EMS)

introduces point mutations by changing G to A

attenuator

section of mRNA transcribed from the leader sequence with domains that can form hairpins and that has two codons for Tryptophan

terminator

specific mRNA hairpin that forms from domains 3 and 4 in the attenuator sequence

prokaryotic genes

primary level = transcription

chromatic remodeling

an important level of eukaryotic gene regulation

histone acetylation

loosens histones from DNA (decreases attraction between basic histone and acidic DNA)

promoter

necessary for transcription initiation at basal level; located immediately upstream of genes often in standard positions, specify transcriptional start site AND direction of transcription (orientation matters), include TATA, GC, and CAAT boxes

enhancers

necessary for maximum level of transcription; time and tissue-specific gene expression, position is NOT fixed, orientation of element doesn't matter

silencers

repress level of transcription initiation; control gene expression in tissue- or temporal-specific ways

high level of expression

induced when cells expose to heavy metals and steroid hormones

transcription factors

bound to enhancers (or silencers) interact with RNA polymerase II and other basal/general transcription factors

isoforms

different proteins produced from same gene; usually tissue-specific and can be found at different stages of development

Dscam gene (flies)

encodes axon guidance receptors that controls neuronal wiring; has most alternative splicing combinations

jumping genes

can move within or between chromosomes causing mutations where they insert

inducible

a product whose production must be stimulated (usually by a particular molecule)

constitutive

a product that is constantly produced

repressible

production that can be turned off (usually by presence of molecule that system is producing)

cis-acting site

regulatory region adjacent to gene whose expression it controls

trans-acting molecules

molecules that binds to cis-acting sites to regulate expression

lacZ

encodes enzyme that converts lactose to usable glucose

lacY

encoded permease needed for transport of lactose into the cell

lacA

encodes enzyme that might play a role in detoxification

operator-binding site

active site

lactose binding site

regulatory site

regulatory site

reversible interaction with lactose controls activity of LacI protein

Catabolite-activating protein (CAP)

• allows lactose metabolism to also be regulated by glucose
• activates expression of lac operon in the absence of glucose
• inhibits expression of lac operon when glucose is present

attentuation

when an RNA hairpin can stop transcription; discovered by Charles Yanofsky looking at tcp operon

cell proliferation

abnormal cell growth and division

metastasis

ability to invade other parts of body

multiple mutations

needed to convert a normal somatic cell into a malignant cell

genomic instability

duplication, loss, and translocation of whole or part of chromosomes

E6

a proto-oncogene; if viral, inactivates human p53 tumor suppressor

RB1

a tumor suppressor; cell-cycle checkpoints, binds E2F; mutation, deletion, inactivation by viral E7; leads to retinoblastoma, osteosarcoma and many other types of cancers;

retinoblastoma

tumor that develops in the retina due to mutations in both copies of the RB1 tumor suppressor gene; is a major cause of blindness

RB

controls the G1 to S cell cycle checkpoint

unphosphorylated RB

inhibits the E2F transcription factor

E2F

induce expression of >30 genes that control G1 to S transition

oncogene

mutant version of a proto-oncogene

ras proto-oncogenes

mutated in 30% of cancers

human E6/E7 proteins

inactivate two human tumor suppressor proteins p53 and RB at specific times during cell cycle

viral E6/E7 proteins

act as gain-of-function oncogenes; they inactivate human p53 and RB all the time, leading to unregulated progression of cell cycle

TS genes

control cell-cycle checkpoints

cellular apoptosis

cell suicide (if chromosomal defects detected)

ras proto-oncogenes

normal function to transmit signals from cell surface to nucleus that tell cell to divide

gain of function mutants

prevent Ras protein from reverting from active to inactive state ras oncogenes

human papilloma virus (HPV)

a human oncogenic DNA virus; many strains cause warts

p53 tumor suppressor gene

is mutated in more than 50% of human cancers

p53 transcription factor

stops cell cycle until DNA repaired and activates genes leading to apoptosis

plasmid DNA preps

can use E. coli to harvest many copies of recombinant DNA plasmids

restriction enzymes

isolated from bacteria (they use to protect against viral infection); recognize a specific ds DNA sequence (many are palindromic)

plasmid vectors

vehicle for carrying and replicating DNA; replicated independently from host chromosome; has set of restriction sites for inserting gene of interest

selection strategies

antibiotic selection or color selection

antibiotic selection

can use antibiotics to selectively grow E. coli cells with particular plasmid

color selection

some plasmids have this to visualize which do (white) and don't (blue) have gene insertions

lacZ gene

is used for blue/white color selection of recombinant plasmids

X-gal

a lactose analog

lactose analog

produces galactose and a blue compound when cleaved by beta-galactosidase

plasmid DNA

a small circular piece of DNA endogenous to bacteria; independent of the main bacterial chromosome and are independently replicated within the bacterial cell

cDNA

copy DNA; exon sequences only

exons

the DNA segments of a gene that contain the sequences that, through transcription and translation, are eventually represented in the final polypeptide product

introns

An intervening sequence of DNA that lies between coding regions in a gene. Are transcribed but are spliced out of the RNA product and are not represented in the polypeptide encoded by the gene

recombinant DNA

contains cDNA of your favorite gene and can be amplified after it has been transformed into a bacterial cell

restriction enzymes/endonucleases

specialized proteins that recognize specific DNA base pair sequences and cut the DNA at those sites by breaking the phosphodiester bonds in the DNA backbone

recognition sites/sequence

specific DNA base pair sequences

sticky ends

unpaired bases after palindromic sequences are cleaved where the cDNA insert is pasted into a digest plasmid vector using the enzyme DNA ligase

DNA ligase

the enzyme used to paste cDNA insert into a digested plasmid vector; uses energy from the hydrolysis of ATP to make the new phosphodiester bonds, resulting in a circular recombinant plasmid DNA

vector

Vehicle for carrying and replicating DNA

purpose of genetic cloning

to produce multiple copies of your favorite gene in order to study the structure and expression of that gene

multiple cloning site (MCS)

a region that most plasmids used for cloning have been genetically engineered to have

lacZ gene

codes for a protein that converts the colorless artificial substrate, X-gal, to a blue colored derivative; the presence of this gene in the plasmid allows for the selection of transformed bacterial cells that have taken up recombinant plasmid as opposed to plasmid only

PolA1

strongly reduces ability to repair DNA; mutation

I, II, III

Polymerase _______ exhibit 5' to 3' polymerization

I, II, III

Polymerase _______ exhibit 3' to 5' exonuclease activity

I

Polymerase _______ exhibit 5' to 3' exonuclease activity

3' to 5'

_______ exonuclease activity used in proofreading

III

Polymerase _______ carries out main task of DNA replication

I

Polymerase _______ is the most present

5' to 3'

elongation of DNA is ______

polymerase

_______ fills in gaps of DNA

ligase

_______ connects pieces of DNA

telomerase

_______ fills in gaps of DNA by 3' end

telomerase

discovered by Blackburn and Greider

endonuclease

________ introduces nicks before and during recombination

heteroduplex

double stranded molecule of DNA produced from genetic recombination

Holliday structure

________ forms during recombination

ligation

________ creates recombinant duplexes

19000-73000 microns

length of one human chromosome

2 meters

length of combined DNA in one cell

5-10 microns

diameter of nucleus

positively

histone is _______ charged.

nucleosome

DNA wrapped around histone proteins

heterochromatin

silent (condensed) chromatin

euchromatin

active chromatin

Acetylated

_______ histones are associated with open form of chromatin

Deacetylated

________ histones are associated with a closed form of chromatin

methylation

________ correlates with genes being switched off; silencing genes

20

number of amino acids

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