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524 Cards in this Set
- Front
- Back
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B form of DNA
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standard form
bp 90° angle to axis one turn--> 10.5 bp bp distance --> 0.34 nm right turned major and minor groove clearly distinctable |
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A form of DNA
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present in dehydration
bp 70° angle to axis one turn --> 11 bp bp distance -->0.26 nm right turned |
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Z form of DNA
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ZickZack form of backbone
left turned helical structure angle between 2 bases much larger |
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Endonuclease
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DNA degradation by hydrolysation of phosphoestherbonds
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Examples of Endonucleases
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DNase 1 (pancreas) ss and ds
DNase 2 (thymus) ss and ds Endonuclease 1 (e.coli) ss and ds Endonuclease 2 (e.coli) AP endonuclease S1 Endonuclease (Aspergillus) ss |
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Exonuclease
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DNA degradation from ends of ss or ds
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Types of Exonucleases
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Exonuclease 1 (e.coli) 3'-5' ss
Exonuclease 2 (e.coli) 3'-5' DNA correction from DNA pol 1 Exonuclease 3 (e.coli) 3'-5' ds Exonuclease 4 (e.coli) 3'-5' ss Exonuclease 5 (e.coli) 3'-5' and 5'-3' rec BC nuclease T7 Exonuclease (phage T7) 5'-3' ds |
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Restriction nuclease
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Destroys foreign DNA
Recognizes specific repetetive sequences in phages Prokaryotic structures prevented from degradation due to methylated A and C produces sticky and blunt ends |
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Types of Restriction nucleases
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Type 1 recognizes defined sequences but cuts apart from them and randomly
Type 2 cuts insiderestriction sequence Type 3 cuts 20-25 nucleotides apart from restriction sequence |
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nucleotide
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Base + sugar backbone + phosphategroup
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nucleoside
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Base + sugar backbone
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pyrimidine base
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Cytosin, Thymine, Uracil
6 ring structure |
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Purin
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Adenine, Guanine
5 ring or 6 ring structure |
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sense strand
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positive strand
coding strand of DNA identical to mRNA |
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antisense strand
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negative strand
noncoding strand of DNA complementary to mRNA as it serves as template |
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Transposons
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moveable genetic elements
in Pro and Eukaryotes can move within one or btw two DNA molecules (Transposition) requires transposase |
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plasmid
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extrachromosomal DNA
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histone like proteins
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hold together supercoils of prokarytic DNA
bacterial binding protein |
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Topoisomerases
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influence tight and loose binding of DNA
important in initiation of replication, fork movements and untangling finished chromosomes after replication |
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Gyrase
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Type 2 topoisomerase
introduces negative supercoils at oriC nicking and closing of 1 strand |
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operon
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polygenic DNA strand
functional sequence of several genes |
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promotor
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recognition sequence for RNA polymerase in prokaryotes
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operator
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binding site for repressor proteins
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sigma factor
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regulator for several genes even on different locations in the genome
when encoded--> protein translation is enhanced --> protein is more resistant to heat |
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Episome
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plasmid that can integrate into chromosomal DNA
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F plasmid
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fertility plasmid of e.coli
about 100 genes when extrachromosomal--> F+ cell whne intrachromosomal--> Hfr cell no F plasmid--> F- cell integration into chromosome at IS elements pilus forms cytoplasmid bridge |
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conjugation
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cytoplasmid bridge
--> 1 strand opened at oriT endonucleoticly --> transferation of 5' end into recipient cell --> transcrition of tra Gene --> complementation to double strands in both cells one way only donor--> male recipient--> female |
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reverse gyrase
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uncoils the positive supercoiled DNA of archea
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DNAB
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helicase
seperates DNA double strands or reannealed RNA strands under consumption of ATP |
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DNAC
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delivers helicase to DNA template
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DNAA
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binds to relaxed or ONLY negative supercoiled DNA
replication initiation factor promotes unwinding of DNA at oriC |
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consensus sequence
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sequence that reflects the most common choice of base or amino acid at each position
mostly A-T in the origin |
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ARS
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autonomously replicating sequence
contains the origin of replication in yeast when mutated --> no replication |
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single strand binding protein
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prevents reannealing during replication
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DNAG
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RNA polymerase primase in e.coli
synthesizes a short primer so DNA polymerase can proceed |
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origin of replication
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required for DNA replication
Starting point for DNA polymerase |
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pre priming complex
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DNAB clamps around single strand
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Ligase
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fuses nicks in DNA
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DNA polymerase 3
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catalyzes nucleotide addition in replication
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DNA polymerase 1
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removes RNA primers and fills the gaps
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Topoisomerase 1
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maintains proper helical density
removes supercoils breaking and rejoining double stranded DNA by cutting 1 strand |
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homologous recombination
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exchange of nucleotides between similar or identical DNA molecules
double strand breaks --> repairing harmful breaks --> genetic variation in horizontal gene transfer |
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horizontal gene transfer
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exchange of genetic material without being offspring
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vector
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vehicle to transfer foreign genetic material into another cell
plasmids virusses cosmids artificial chromosomes |
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telomerase
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enzyme that adds DNA sequence repeats to the 3' ends at telomer regions
only in eukaryotes |
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Rolling circle
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nick at origin
5' end displaced and covered by ssb 3' primer for DNA synthesis Replisome attches nd okazaki fragments are formed |
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Alternative splicing
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different ways of putting together exons
including or excluding single exons contributes to genetic variation in 95% of all human genes ONLY misregulation can lead to disease |
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shine dalgarno sequence
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ribosome binding site
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ribosomal catalytic site
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place of peptide bond formation
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ribosomal p site
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reading site
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superoxide dismutase
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antioxidant
enzyme that catalyzes the dismutase (redox-->2 outcomes) of superoxide o2- to oxygen and hydrogen peroxide SOD1 in cytoplasm SOD2 in mitochondria SOD3 extracellular |
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Radiation
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breaks covalent bonds in DNA (ss& ds)
leading cause of chromosome mutation kills cells at high doses (cancer therapy) |
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UV
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causes pyrimidines (U,T and C) to form abnormal dimer bonds --> kinks --> no replication
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photolyases
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absorb energy from visible light (so doesnt work in the "dark"
breaks pyrimidinic dimers --> DNA repair only for UV damage |
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alkyl transferase
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removes alkyl groups from o6 of guanine (majr carcinogenic lesion in DNA)
no real enzyme, because not regenerated system can be saturated |
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MGMT
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Methylguanine Methyltransferase
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mismatch repair
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corrects errors of replication
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Excision repair
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corrects damage to template
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Nucleotide excision repair
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in humans 27-32 nucleotides removed
in bacteria: very short patch few nt; short patch 12-13 nt; long patch 1500-9000 endonuclease cleavage up and downstream DNA polymerase 1 --> further degradation and replacement of DNA DNA ligase --> seals nick defects in NER--> xeroderma pigmentosum, cockayne syndrome, trichthiodystrophy removes benzopyrene-guanine adducts (smoking) removes thymine psoralen and guanine-cisplatin adducts (chemotherapeutic drugs) |
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Trichthiodystrophy
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autosomal recessive
point mutation intellectual impairment brittle hair |
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cockayne syndrome
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autosomal recessive
point mutation growth failure impaired development of nervous system abnormal sensitivity to sunlight premature aging |
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xeroderma pigmentosum
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autosomal recessive
point mutation UV light damage to DNA cant be repaired --> excessive skin cancer accelerated neurological degeneration 7 different types |
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base excision repair
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removes small, non helix distorting base lesions
initiated by DNA glycosylases (removes base)--> AP site AP endonuclease creates nick in the phosphodiester backbone of AP site more complicated than NER |
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DNA glycosylase
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8 different forms in humans
diffuse along minor groove until they find a lesion then remove base --> AP site |
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Deletion
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DNA loops out on template strand
--> DNA polymerase skips base --> deletion occurs |
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Addition
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DNA loops out on new strand
DNA polymerase adds untemplated bases |
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wobble base pairing
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uncorrect base pairing leads to mutation in second generation progeny
either transversion or transition reasons: tautomerism, depurination, deamination, base analogs, hydroxylation, alkylation |
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bromouracile
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behaves like thymine in normal state
behaves like cytosine in rare state --> transition mutation |
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base modifying agents
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nitrous acid (deamination)
methylmethanesulfonate hydroxylamine |
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intercalating agents
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thin, plate like hydrophobic molecules
insert themselves into adjacent base pairs --> frameshift mutations e.g. proflavin, ethidiumbromide |
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DNA repair mechanisms
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Photoreactivation
repair of AP sites proofreading NER BER recombinational repair sos repair |
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sos repair
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error prone
sos genes repressed by lexA (binds to 20 bp consensus sequence in SOS box DNA damage activates SOS genes due to accumulation of ssDNA at replication fork --> recA becomes activated and interacts with lexA --> lexA cleaved from repressor--> SOS gene expressed |
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AP sites
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apurinic/apyridinic site
ca. 10.000 produced /day/cell intermediate in BER if unrepaired--> mutation because fork stalling-->switch to translesion synthesis |
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translesion synthesis
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DNA polymerase switches to translesion polymerase
--> insertion of bases opposite damaged nucleotides facilitated --> or bypassing damaged NT --> higher property of wrong base |
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Li Fraumeni Syndrome
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great susceptibility to cancer
SBLA syndrome (synonyme) germline mutation of p53 inherited de novo in embryogenesis or from germ cells autosomal dominant |
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phenotype
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characteristics
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genotype
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genetic nature
combination of similar or different alleles of 1 gene |
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defect mutants
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species defective in their ability to use certain substrates
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resistant mutants
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species resistant to some antibiotics
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mutation rate
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mutation probability per time
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mutation frequency
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number of mutations per population
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replacement substitution
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point mutation in first and second place non synonymous, in third often silent
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luria delbrück experiment
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mutations occur in absence of selection mechanisms, not as reaction of circumstances
mutation was spontaneous--> unequal distribution |
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genetic assimilation
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phenotype produced in response to environmental conditions becomes genetically encoded (EPIGENETICS)
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allele
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one of several forms of the same gene
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haplotype
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genotype for a sequence of linked allelson a chromosome that are inherited together
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types of mutations
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point mutation
gene duplication transposable elements chromosome inversions chromosome translocations polyploidity |
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point mutations
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insertion or deletion leads to frameshift
can be silent (same aa) missense (other aa) nonsense (stop codon) neutral ( aa with similar properties) --> can lead to transition or transversion example: sickle cell anemia |
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sickle cell anemia
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point mutation in hemoglobin gene
only 1 bp of 146 aas mutated (CTT-->CAT) heterozygous advantage in malaria regions leads to anemia and obstructed capillaries ( --> pain, organ damage) |
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open reading frame
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from start to stop codon
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suppressor mutation
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second mutation that reverts the phenotypic effect of the first but happens at different sites
can forward wild type to mutant and vice versa genes often encode tRNA --> recognize stop codon and insert an aa each stop codon has its suppressor they compete with release factors |
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mutation rate influenced by
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proofreading
mismatch repair fast DNA polymerase --> less thoroughly |
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overprinting
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new start codon within ORF
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new alleles come from
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point mutation
crossing over overprinting posttranslational changes reverse transcription of mRNA |
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variety of HIV genes
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due to posttranslational changes
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gene duplication
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can cause mutation
important in evolution as the additional copy is free from selective pressure --> new genes duplication of oncogenes leads to cancer |
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gene family
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cluster of related genes
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pseudogene
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untranscribed gene
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polyploidy crops
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induction by colchidine during meiosis
--> larger, grow faster |
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colchidine
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spindle poison
stops cell division in metaphase |
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plant hybrids
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tetraploid crossed with diploid --> plant is sterile --> seedless
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parthenogenic
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reproduction without fertilization
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autopolyploidity
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union of gametes of the same species
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hybridization
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union of gametes of different species
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aneuploidity
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los or gain of one or more through nondisjunction chromosomes in mitosis or meiosis
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monosomy
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loss of 1 chromosome 2n-1
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trisomy
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gain of 1 chromosome 2n+1
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nullisomy
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loss of 1 chromosome pair 2n-2
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disomy
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addition of 1 chromosome in a haploid n+1
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turners syndrome
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absence of sex chromosome xo in females
leads to sterility, short stature, amenorrhea |
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klinefelters syndrome
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xxy in males
leads to lower muscle tone in growings, less facial and body hair, increased breast tissue, low testosteron |
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crossing over
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exchange of genetic material between homologous chromosomes
in prophase of meiosis 1 creates nonparental genotypes and phenotypes for linked genes--> new alleles can cause problems. aberrations in individual chromosomes like deletion and duplication(imbalanced) or Inversion or translocation (balanced) |
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classes of alleles
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amorphous --> complete loss of function
hypomorph --> reduction of function hypermorph --> increase of function neomorph --> gain of new function |
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pseudogenes
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nonfunctional genes
arise by duplication (nonprocessed) or by retrotransposition ( processed) |
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processed pseudogenes
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mRNA is retranscribed into DNA and integrated into the genome
characteristics --> small direct repeats poly a tail |
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reciprocal translocation
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pieces of seperate chromosomes breaking off their original chromosomes and switching places to produce a derivative chromosome
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deletion mapping
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technique to ascertain location of mutations
deletion is verified if it fails to recombine with point mutationsin the same gene |
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paracentric inversion
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dont include centromer
broken only in 1 arm |
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pericentric inversion
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include centromers
both arms broken |
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robertsonian translocation
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nonreciprocal whole arm translocation of acrocentric chromosomes
cause of fimiliar down syndrome |
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deletion
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mostly lethal
cri du chat terminal (at the end) or interstitial |
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acentric fragment
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no centromer through crossing over within a paracentric inversion
lethal |
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dicentric bridge
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chromosome with 2 centromers
breaks randomly apart |
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balancer chromosome
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recombination would lead to acentric or dicentric chromosome --> lethal
product of inversions used as tool to prevent crossing over to keep for example heterozygous mutations in lab |
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ames test
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test for mutagenesity of chemicals
1. his - auxotroph s.typhimurium + rat liver enzymes (met. products) on plate with chemical +small amount of histidine --> bacteria grow and event. mutate 2.when mutated bacteria keep growing 3. 48 h incubation |
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site specific mutagenesis
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mutant alleles are synthesized and transformed into cell cultures to investigate proteins or use changed protein behaviour in industry ( washing powder)
DNA primer with mutation --> hybridize with DNA |
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Nucleoplasm
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chromosome/ chromatin containing region in the nucleus
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chromatin
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chromosome + protein (1:2)
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nucleosome
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packaging of DNA into 11 nm beads on a string (histones)
not present in promoter regions |
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solenoid
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highly condesed in interphase with interspersed loops
in metaphase transcriptionally silent 20000-100000 bp |
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segregation of robertsonian translocation
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25% lethal
25% sick 25% healthy 25% carrier |
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histones
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positively charged
H1,2*( H2a, H2b, H3, H4) form nucleosome acetylated or methylated at lysine--> change of chromatin structure |
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acetylation of histone lysine
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by histone acetyl transferase
--> chromatin condensation |
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methylation of histone lysine
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by histone methyl transferase
--> chromatin highly compacted |
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Histone H1
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"clamps" the DNA strand to the core of the histone (octamer)
binds to linker DNA |
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solenoid
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coiled 11 nm fibre pulled into solenoid
no gene transcription |
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Eu chromatin
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less condesend
10 % active genes present in interphase |
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Hetero chromatin
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highly condesed
low transcription can cause gene silencing present in interphase |
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Telomers
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tandem repeats rich in g
nobel prize for Elizabeth Blackburn, Carol Greider, and Jack Szostak in 2009 normally 3' overhang (300bp) that forms T loop and can form G=G (guanine quarters) |
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short interspersed repeated sequences
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SINE
100-500 bp DNA representing reverse-transcribed RNA molecules originally transcribed by RNA polymerase III into tRNA |
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long interspersed repeted sequences
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LINE
>5000 bp transcribed to an RNA using an RNA polymerase II promoter that resides inside the LINE code for the enzyme reverse transcriptase, and many LINEs also code for an endonuclease |
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microsatellites
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short tandem repeats in DNA
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telomerase
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special reverse transcriptase
adds bp to 3' end after RNA template on telomerase itself --> lenghtening of telomers |
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transcription in prokayotes
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promotor --> -10 (tata or pribnow) to -35
RNA polymerase binds to promotor |
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lac operon
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when lactose absent --> repressor binds
lactose --> inducer -->binds to repressor which is then released NEGATIVE regulation IPTG can also act as inducer |
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merodiploid
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has some genes twice
they are in cis when found on same DNA molecule they are in trans when they are found on different DNA molecule |
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Oc allele mutation
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cis dominance --> whichever alleles is in cis to Lac Z+ is dominant
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lac I
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codes for lac repressor
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lac Z
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codes for galactosidase
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lac y
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codes for lactose permease
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allosteric regulation
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is the regulation of an enzyme or other protein by binding an effector molecule at the protein's allosteric site zB inducer binds to repressor and prevents it binding to operator
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Catabolite activating protein
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turns on lac gene in absence of glucose
activated by binding to camp in condition of low glucose complex binds to promotor --> increased transcription POSITIVE control |
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camp
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second messenger in cellular signal transduction
activation of proteinkinases in bacterial signal pathway for lack of glucose |
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aporepressor
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repressor + corepressor
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trp operon
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regulated by repression
NEGATIVE control low tryptophan--> no repression high tryptophan --> repression end product inhibits the operator also regulated by attenuation |
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Attenuation
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negative control, further decrease of transcription
only in prokaryotes, as simultaneous translation and transcription 4 parts in first sequence that can form hairpins that prevent each other 2+3 anti termination 3+4 termination |
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non dividing cells
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nerve tissue
muscle cells red blood cells |
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restriction point
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point in g1where cell becmes comitted to further cell cycle
controlled by cyclin D |
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cell cycle
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interphase : g1 + s phase + g2 and
mitosis: prophase, metaphase, anaphase, telophase, cytokinesis |
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meiosis
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reductional division--> mitosis 1
germatogenesis --> mitosis 2 yields in 4 haploid germ cells |
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Interphase
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G1 --> check for cell growth, preparation for cell division
S Phase --> synthesis of DNA --> homologous chromosomes G2 --> check for cell growth, propper replication, preparation for cell division, organelles replicated, molecules for cell division synthesized |
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G2
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check for cell growth, propper replication, preparation for cell division, organelles replicated, molecules for cell division synthesized
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parthenogenesis
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asexual reproduction in which an unfertilized egg developes into an offspring ( stick insects)
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oogenesis
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stem cells have already started differentiating in embryonal state--> while diploid
1 gamete produced per meiosis--> 3 barr bodies, 1 egg all chromosomes in recombination during prophase 1 |
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crossing over
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mixing of homologous chromosomes in prophase 1 (diplonema)
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spermatogenesis
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stem cells not yet differentiating
4 gametes per meiosis begins in puberty differentiation while haploid sex chromosomes excluded from recombination in prophase 1 |
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sertoli cells
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nourish developing sperm
produce part of seminal fluid establish blood testis barrier |
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leydig cells
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secrete testosterone
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organelles formed in spermatogenesis
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acrosome (enzymes for ovum breakdown)
middles section ( with mitochondria) axonome (cilia and flagella) cell surface cytoplasm reduction |
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regulation of gene expression
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eukaryotes: promotor --> transcription factors bind
prokaryotes: operon --> repressor and inducer bind |
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polynucleotide kinase
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adds phosphate to 5' OH
--> labelling --> permit ligation |
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alkaline phosphataes
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removes terminal phosphate from 5' or 3'
prevents reannealing enables radioactive labelling |
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disadvantage of genetic engineering
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different sources (zB bacteria and human) protein modification can be altered) due to wrong folding of eukaryotic DNA
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production of rDNA
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1. cutting with same restriction enzyme
2. pasting DNA together 3. insert into bacteria and clone 4. screening to identify clone |
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6 base cutters
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eco r1 or bamH1
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plasmid features
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origin of repliation (always top)
selectable marker unique restriction site |
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types of vectors
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plasmid
phage cosmid shuttle BAC YAC (naked DNA) |
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cos side
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cohesive side in phage lambda
produces sticky ends makes plasmid circular -> cosmid |
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cDNA
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DNA synthesized from mRNA using reverse transcriptase and DNA polymerase
used to clone eukaryotic genes in prokaryotes ( no introns) produced naturally by retrovirus (HIV1/2) |
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adapter
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short chemically synthesized double stranded DNA
links end of 2 DNA molecules adds sticky ends to DNA --> can be ligated into plasmid no risk that restriction site is in vector |
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shuttle vector
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can prpagate in several host types
mostly plasmids replicate autonomously or integrate into host DNA used to transport genes between different organisms like plants--> animals |
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genes causing cancer when mutated
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growth factors
receptors signalling pathways repair systems apoptosis factor ras cellcycle regulation (p53) |
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restriction map
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map of known restriction sites
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tryptophan starvation
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leader peptide has two adjacent trp residues --> ribosome stalls
2-3 loop forms |
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puc 19
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high copy number
polylinker in lacZ marker is amp detection by x-gal --> turn blue in presence of betagalactosidase --> blue --> not inserted white --> inserted |
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phage lambda vector
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central region of the chromosome is cut out ans replaced with 15 kb insert
inserted DNA plus vector DNA is packed into heads --> infecting e.coli --> lytic cycle large number of restriction sites |
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cosmid
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packaged into lambda phages , intorduced in e.coli
(45 kb) dont occur naturally |
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YAC
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cloned in yeast
telomere and centromere autonomously replicating for very large fragments (2000 kb max) |
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BAC
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from F plasmid
very stable 200 kb max |
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ethidium bromide
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--> frameshift mutation
--> mutagenic |
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SOS repair
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error prone
genes expressed by lexA activated by ssDNA cumulation at fork |
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DNA polymerase 1 in e.coli
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fills gaps in duplexes by addittion of nt to 3'
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terminal transferase
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adds homopolymer tails to 3' OH of linear duplexes
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exonuclease 3
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removes nt from 5' to expose 3' ss ends
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human genome
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22000-30000 genes
only 1-1.5 % coding proteins 50 % repetetive DNA |
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gene families
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several similar genes
evolve by duplication share nucleotide or protein sequence |
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tamdemly arranged genes
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gene cluster created by tandem repeats
--> same gene several times in a row zB in rRNA--> produced much quicker due to high need for it |
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repetetive DNA
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only in eukaryotes
functional like tandemly arranged genes or telomers no known function like centromers, VNTRs (mini satellites) |
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restriction fragment length polymorphism
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insertion or deletion of restriction site
used for assaying point mutation like sickle cell detectable on gel |
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segmented genomes
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replication in cytoplasm
RNA polymerase produces monocistronic mRNA from each segment in para/orthomyxoviridae |
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concatemers
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long continuous DNA molecule that contains multiple copies of the same DNA segments linked in series
at the end of linear DNA recognized by endonucleases |
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mimivirus
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large size (like small bacteria)
gramstain positive genes for nucleotide and amino acid synthesis no genes for ribosomal proteins |
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viruses
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either RNA or DNA genome
no ATP or protein production (except mimivirus) first described 1798 by jenner ( all pathogenic organisms= viruses) |
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zoonosis
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non human viruses like ebola or hanta (inhaling mice urin)
infect humans accidentally very lethal |
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variolation
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taking drieb scabs of smallpox victims, powder them and blowing into nose to achive immunity
established in china |
|
|
meselson and stahl
|
semi conservative replication of DNA
|
|
|
griffith avery mc carthy mc leod
|
DNA can transform bacteria from 1 tpe into another (s and R strain)
|
|
|
hershey abd chase
|
DNA is infectious agent (bacteriophage)
|
|
|
watson crick
|
double helix, x ray diffraction photo of DNA
|
|
|
rolling circle
|
1. nick at ori c
2. 5' end displaced 3. 3' end serves as primer--> duplication of strand that stays 4. nicked strand + RNA primer + DNA poly 3 = okazaki fragments in many phages ( lambda) |
|
|
viral + sense ssRNA
|
directly infectious
5' UTR CAP and poly A tail zB picorna ( HAV; polio, common cold); Flaviviridae (HCV) |
|
|
viral - sense ssRNA
|
not directly infectious
brings own polymerase that produces + strand (mRNA) --> replicative intermediate doublestrand |
|
|
DNA replication
|
2 forks--> bidirectional
|
|
|
gutless vector
|
foreign DNA into mammalian cells
derived froma adenovirus cut out almost everything--> non toxic can carry 36 kb DNA |
|
|
production of genomic library
|
complete digestion (enzymes with plenty restriction sites)
partial digestion (enzymes with few restriction sites) mechanical shearing |
|
|
clade
|
group of organisms derived from one ancestor species
shared characteristics |
|
|
human T cell leukemia virus
|
retrovirus
causes adult T cell leukemia infects CD4 positive cells gag pol env + 2 LTR's |
|
|
oligonucleotide
|
synthetic DNA strand used for cDNA screening
|
|
|
cDNA screening
|
library members translated, protein on membranes
labelled probe on membrane hybridization washing |
|
|
speration of chromosomes
|
by flow cytometry (stain, detection with laser)
|
|
|
isolation of mRNA
|
column where 3' poly A tail stick to
--> seperation from rRNA, tRNA, small RNA |
|
|
poduction of cDNA library
|
isolation of mRNA
poly T primer to poly A tail reverse transcriptase creates RNA DNA hybrid RNase H degrades mRNA almost completly, leaving primers DNA poly 1 synthesizes 5'-3' and removes primers DNA ligase --> ds cDNA |
|
|
southern blot
|
to detect DNA of interest
1. restriction enzyme cleaves DNA 2. electrophoresis -->separation according to size 3. blotting on membrane with alkaline solution 4.hybridization with labelled complementary sequence 5. wash 6.x-ray --> detection of gene of interest |
|
|
northern blot
|
like southern, but RNA instead of DNA
mRNA --> smear rRNA --> 1 big, 1 small tRNA--> cloudy band on bottom small RNA --> not visible, because they cant take up enough dye |
|
|
western blot
|
like southern but proteins
|
|
|
DNA sequencing
|
notation of the exact nt sequence of a DNA strand
maxam gilbert (chemically) sanger (enzymatic) |
|
|
sanger method
|
DNA chains of various length, labelled dideoxynucleotide at the end
nowadays labelled with 4 fluorescent dyes and scanned with laser |
|
|
dideoxynucleotide
|
has no 3'OH --> last nucleotide
|
|
|
maxam gilbert
|
DNA cleavec chemically --> depurination of purins, methylation of pyrimidines
5' end labelled used for small molecules |
|
|
pyrosequencing
|
used for very small molecules
1. primer bases added, when basepair forms--> pyrophosphatase released --> converted to light by sulfurylase (ATP intermediate) and luciferase when no basepair forms--> nt degraded by enzyme apyrase used for epigenetic sequencing as bisulfite treatment converts unmethylated C to T --> ratio of T and C gives information about methylation of CpG islands |
|
|
SOLID technology
|
for very little pieces of DNA
walking promer oligonucleotide ligation adds always 2 bases |
|
|
restriction point
|
at end of g1
regulated by MPF --> cdk2,4,6 cyclin D; cdk 2 cyclin E |
|
|
Hartwell, Nurse, Hunt
|
nobelprize 2001 for detection of key regulators of cell cycle
|
|
|
Roo Johnson experiment
|
fusing 2 cells in different cellcycle state--> signals influencing each other --> G2 stopped by influence of M and S Phase
|
|
|
methylation
|
protection of bacteria
keyplayer in epigenetics gene regulation in tumors density of chromosomes proofreading activity |
|
|
MPF
|
maturation promoting factor
complex of cyclin and cdk only formed in mitosis when cyclin level is highest cdk activated by dephosphorylation different MPFs |
|
|
MPF tasks
|
phosphorylation of proteins (lamin--> nuclear envelope breakdown)
regulation of M--> G1 --> cyclin B degradation regulation of spindle apparatus phosphorylation of H1 histone |
|
|
MAPK
|
3 steps, 2 phosphorylations
regulation of proliferation, gene expression, mitosis, apoptosis and many others stress, inflammation etc.--> signal reduced, inhanced |
|
|
p 53
|
guardian of the cell
tumor suppressor activated by DNA damage, cell cycle abnormalities, oxidative shock, osmotic stress without p53-> no apoptosis--> cancer activates DNA repair activates miRNA induces cell cycle arrest at G1and G2 when DNA is damaged by stimulating the synthesis of inhibitors of cyclin-dependent kinases, such as p21--> inhibiting RB releasing E2F transcription regulator in damaged cells activated by phosphorylation of N-terminal labelled by mdm2 for proteasome overexpression--> no function |
|
|
Li Fraumeni Syndrome
|
inheritation of only one functional p 53 gene
often cancer in early adulthood |
|
|
HPV and cancer
|
early genes interact with tumorsuppressors of the cell
E6 with p53 and E7 with RB --> downregulation --> proliferation of the cell --> warts in HPV 16 and 18 --> cancer development in cervix ( carcinoma in situ) |
|
|
RB
|
retino blasoma tumor suppressor gene
regulates restriction point named after condition in early childhood when gene mutated when activated --> no progression of cell cycle deactivation by phosphorylation by Cyclin D/CDK4/CDK6, followed by additional phosphorylation by Cyclin E/CDK2 (RESTRICTION POINT) --> release of E2F |
|
|
cell cycle progression
|
stimulating --> mitogens, cyclin kinases
inhibiting --> external inhibitory signal (TGF beta), DNA damage |
|
|
lack of proofreading
|
DNA poly 1
RNA viruses retroviruses |
|
|
immortalization of cells
|
indefinite proliferative life span
every cancer cell is immortalized |
|
|
transformation of cells
|
no response to normal regulators
(2nd meaning: uptake of foreign DNA by bacteria) 1. initial event 2. immortalization 3. inhibition of apoptosis |
|
|
2 step carcinogenesis
|
transformation
(promotion) progression HNPCC |
|
|
promotion
|
altered DNA repair
accumulation of mutations cell cycle promotion inhibition of apoptosis |
|
|
Progression
|
genomic instability due to several mutations
more mutations irregular expression of apoptosis drug resistance (different to csc) |
|
|
Cancer stem cells
|
certain form of breast cancer associated
csc have the ability to renew themselves and generate all kind of cells --> metastasis and more tumors different signalling pahwas relapse of cancer after some years with no response to drugs (breast cancer --> tamoxifen) |
|
|
loss of heterozygosity
|
SNP's (point mutations) --> heterozygousity
when 1 copy is lost due to mutation --> heterozygousity is lost 2nd mutation--> function is lost When this occurs in tumorsupressor gene --> cancer |
|
|
proto oncogenes
|
genes coding for receptors, cell cycle control, signal transduction, ras, etc.
when mutated --> ONCOGENES |
|
|
BRCA 1
|
tumorsuppressor gene
acts together with BRCA 2 and RAD 51to repair damaged DNA when mutated chance to get breast and ovarian cancer is very high --> angelina jolie |
|
|
nuclear transcription factors
|
jun fos myc rel
|
|
|
angiogenesis
|
formation of new blood vessels
|
|
|
uncontrolled regulatory mechanisms in cancer progression
|
increased proliferation
increased survival suppressed differentiation immortalization decreased apoptosis loss of adhesive properties increased migratory properties |
|
|
oncogenes
|
tumor causing genes
mutated proto oncogenes identified through retroviral pick up and causing cancer in animals mutation in oncogenes --> GAIN of function genetically dominant 1911 1st oncogene identified--> rous sarcoma virus |
|
|
transformation assay
|
isolate DNA from tumor
transfer into mouse cells transformed cells in focus 2nd culture extract DNA |
|
|
Normal cell
|
flat morphology
anchorage dependent density dependent |
|
|
cancer cell
|
rounded morphology
anchorage and density independent |
|
|
ras
|
small GTPase
involved in signal transduction when activated by incoming signal via receptor --> activation of cell growth, differentiaition, survival mutation can lead to permanent expression of ras --> proliferation |
|
|
erbB
|
growth factor receptor
activated tyrosine kinases (--> signal transduction) when mutated --> oftern overexpression --> proliferation |
|
|
viruses associated with cancer
|
RNA --> T Cell Leukemia Virus, HIV (indirect)
DNA --> HPV, Ebstein Barr (BUrkitts lymphoma, nasopharyngeal lymphoma), HBV/ HCV, Kaposis sarcoma associated (tumor of plasma cells) |
|
|
retroviruses and oncogenes
|
terminally redundant sequence R
--> integrate into genome + acting as promoter --> activation of downstream genes on host DNA when retrovirus integrates near protooncogene, can cut it out --> mutate to oncogene --> invading next cell --> proliferation |
|
|
E6 and E7
|
interfere with restriction point by inhibiting p53 (reducing half life) and RB (binding instead of E2F)
early genes (genes for replication) for HPV 16 and 18 |
|
|
HPV
|
in squamous epithelium
associated with warts (6+11) and cervical cancer (16+18) |
|
|
Metaplasia
|
conversion of one type of epithelium to another, zB secretory to squamous due to chronic injury
|
|
|
dysplasia
|
combination of abnormal cytologic appearance and abnormal tissue architecture in early neoplasm
precancerous until travesing the basement membrane |
|
|
cervical cancer
|
CIN 1(mild dysplasia)-CIN3(carcinoma in situ)
CIN 2 cango back to normal, when HPV 16 and 18 neg. screening by PAP smear |
|
|
HBV
|
no integration
chronic liver cell injury--> cancer |
|
|
Ebstein Barr
|
infects lymphocytes
interferes with c-myc herpes family --> burkitts lymphoma, nasopharyngeal carcinoma in middle africans --> kissing disease B cell lymphoma in immunosupressed (AIDS) --> hodgkins disease |
|
|
kaposis sarcoma associated herpes virus
|
commonly associated with AIDS
|
|
|
Transposable elements
|
in prokaryotes (btw. chromosome, plasmid, phage) and in eukaryotes (chromosome(s))
NON homologous recombination important in evolution integrates in genes, regulatory sequences can cause deletion and insertion, translocation |
|
|
Retrotransposons
|
reverse transcriptase makes DNA copies from RNA
copies integrate at new site (eukaryotes) |
|
|
IS elements
|
insertion sequence in prokaryotes
genes for mobilization and insertion encoded flanked by inverted terminal repeats max 5 kb similar to cre lox uses host replication enzymes produces stggered cut at target site + small direct repeats--> change of DNA even after it is cut out again can cause mutation |
|
|
prokayotic composite transposons
|
genes flanked by IS elements
forms lollipop structures stable |
|
|
noncomposite transposons
|
similar to composite but no IS elements
|
|
|
cointegrate
|
molecule produced as intermediate in crossing over recombination of transposable elements (--> 1 big element before seperation again)
|
|
|
temperate bacteriophage MU
|
linear phage DNA + host DNA at the end
integrates into E.coli chromosome |
|
|
barbara McClintock
|
Nobel prize 1983 for transposable elements
purple spots in white corn due to insertion and leaving of transposons into colour gene big spots --> transposon has left early, so more colour could be expressed |
|
|
eukaryotic transposons
|
autonomous (Ac) or non-autonomous (Ds)
Ds needs Ac |
|
|
Ac transposition mechanism
|
only during replication Ac changes location
no copies left behind either to already transcribed site --> no net increase or to not yet replicated site --> net increase |
|
|
Ty in yeast
|
transposable element in yeast
similar to bacterial transposons terminal repeats integrate at non-homologous sites target site duplication share properties with retrovirus ( synthesize RNA--> DNA copy--> integration) upto 70% repetetive sequences |
|
|
Drosophila retrotransposon
|
about 15% of genome mobile in drsophila
2 kinds: copia--< similar to TY P-elements in males + M females --> hybrid degenesis encodes P protein flanked by TIR cut and paste mechanism autonomous |
|
|
hybrid digenesis
|
muation in cell line of D. melanogaster
leads to male sterility --> no progeny P strain males + (lab) M strain females ( no repressor in cytoplasm) --> P strain progeny --> wild type females express piRNA in maternal line --> silencing of germline retrotransposons |
|
|
SINE's
|
short insert of retrotransposons
Alu practically always unique as insertion randomly found in eukaryotic genome (11%) |
|
|
piRNA
|
postrtranscriptional silencing of retrotransposons in germline cells
|
|
|
LINE's
|
long interspersed elements
code for RT no LTR's ORF |
|
|
genome rearrangement from transposons
|
exactly like cre-lox
--> same orientation --> cut out --> different orientation --> flip --> when very close, same orientation --> deletion or duplication |
|
|
gene duplication
|
ectopic recombination (unequal crossing over)
replication slippage retrotransposition aneuploidy polyploidy --> causes illness and/or evolution --> second copy free from selective pressure-> new alleles, new function --> gene families (Hox, immunoglobin, MAPK) |
|
|
Bacterial gene transfer
|
conjugation
transformation transduction transosable elements -->always unidirectional |
|
|
transformatin in bacteria
|
griffith 1928
--> phenotypic change --> antibiotic resistances some pick up naked DNA naturally (b. subtillis) or are engineered (E.coli) electroporation and chemical modification --> induce uptake |
|
|
lederberg and tatum
|
bacterial conjugation
2 auxotroph --> heterotrophs |
|
|
plasmids
|
self transmissible or mobilizable ( need another plasmid)
high copies(20-700) / low copies(1-12) --> number of plasmids per cell |
|
|
promiscious plasmid
|
transfer system allows transfer to unrelated species
|
|
|
F plasmid
|
self replicating
F+ and F- --> donor and recipient complete transfer (1-5 ') --> F- becomes F+ rolling circle mechanism |
|
|
pilus
|
main strctural component--> pilin
recognizes several receptors on host cell |
|
|
conjugation
|
pilus attaches--> close contact--> nick formation--> plasmid opened at ori--> 1 strand dislocated--> duplication
|
|
|
Hfr strains
|
high frequency recombinant strands
F plasmid has integrated into genome by hmologous recombination or transposition attempt to transfer whole genome (100 min--> very rare) recombintion in recipient DNA ( crossing over or transposition plasmid itself not completly transfered bcs starts in the middle azi tan lac gal ORIENTATION VARIES |
|
|
prime factor plasmids
|
leave chromosomal DNA by homologous recombination --> deletion in chromosome --> transfer may produce merodiploids
|
|
|
dNTPs
|
deoxynucleoside triphosphate--> adding of nucleotides
|
|
|
type 1 topoisomerase
|
relaxe DNA by nicking and closing 1 strand of duplex DNA
removing neg. supercoils |
|
|
type 2 topoisomerase
|
change DNA topology by breaking and rejoining double stranded DNA
introduces neg. supercoils |
|
|
primsome
|
helicase + primase
|
|
|
segmented genome
|
2 or more pieces of RNA/DNA packaged in the same particle (orthomyxoviridae)
|
|
|
ds DNA virus
|
Adenoviridae, Poxviridae, Herpesviridae, Papovavirirdae (Hepadnaviridae --> 1 strand longer)
replication in nucleus or cytoplasm |
|
|
ss DNA virus
|
Parvoviridae
replication in nucleus |
|
|
dsRNA virus
|
Reoviridae, Birnaviridae
Segmented genomes |
|
|
ssRNA + sense virus
|
Picornaviridae, Togaviridae, Flaviviradae, Coronaviridae, Caliciviridae, Retroviridae (diploid + RNA)
|
|
|
ssRNA - sense virus
|
orthomyxoviridae, Rhabdoviridae, Paramyxoviridae, Filoviridae, Arenaviridae, Bunjaviridae,
segmented or not segmented |
|
|
tRNA arms
|
Acceptor arm (aa)
Anticodon Arm (ac) TC arm (pseudouridin) D arm (dihydrouridine) |
|
|
ribozyme
|
RNA molecule capable of catalyzing specific biochemical reactions
|
|
|
helicase
|
unwinds DNA double helix
|
|
|
Evans, Cappeci, Smithies
|
Nobel Prize 2007 for Knockout mice
|
|
|
chargaff
|
A pairs T, G pairs C
|
|
|
multipartite genome
|
segmented viral genome packed in several particles that invade a cell together
|
|
|
cos side
|
12 nt sequence at end of linear viral chromosome in phage lambda--> rolling circle mechanism
|
|
|
terminal redundancy in phages
|
form concatamers in linear DNA --> cutting site for endonuclease
|
|
|
Hoogstein arrangments
|
unusual bp to stabilize tRNA
|
|
|
Fire, Mello
|
nobelprize 2006 RNAi
|
|
|
RNAi
|
silencing of gene expression induced by siRNAs
|
|
|
micro RNA
|
ss RNA processed by RNAse3 nuclease from 70bp hairpin structures
interfere with mRNA --> silencing or destruction activated u.a. by p 53 |
|
|
sh RNA/ short hairpin RNA
|
sense + antisense + loop in between them
forms hairpin due to complementary of sense and antisense precursor if siRNA --> incorporation into RISC |
|
|
nucleotide excision repair
|
removes 27-32 nt in euk.
removes upto 9000 nt in prok. (long patch) detects bulky lesions and removes them replacement synthesis by poly 1 |
|
|
Base excision repair
|
wrong base detected by glycosylase
AP endonuclease cuts backbone DNA polymerase synthesizes new nt ligase seals nick |
|
|
mismatch repair
|
repairs looping outs on new( --> insertion) or old strand (deletion)
different in euk. and prok. DNA polymerase synthesizes new nt ligase seals nick associated with HNPCC |
|
|
depurination
|
A or G replaced with random base
|
|
|
base analogs
|
similar to normal base
sometimes mutagenic aciclovir |
|
|
intercalating agents
|
thin platelike hydrophobic molecules
insert themselves BETWEEN bases --> mutagenic |
|
|
recombinational repair
|
damaged portion of DNA is not replicated
--> gap cant be accepted --> incooperation of complementary sequence of normal copy |
|
|
wild type
|
original species
|
|
|
auxotroph
|
require additional growth supplement
|
|
|
defect mutants
|
defective in their ability to use certain substrates
|
|
|
resistant mutants
|
resistant to certain antibiotics
|
|
|
induced pluripotent stem cells
|
yamanaka 2006
2012 --> nobel prize genes for transcription factors in cells via viral vector --> c myc, Oct 4, Klf 4, Sox 2 can cause cancer due to viral vector new method--> lowering pH, some cells survive and express Oct 4 |
|
|
haplotype
|
genotype for a suite of linked alleles on a chromosome
|
|
|
mutation rate
|
propability of particular mutation per unit time
|
|
|
mutation frequency
|
number of times of a particular mutation in a population
|
|
|
types of mutations
|
point mutation
gene duplication transposons chromosome inversion or translocation polyploidy |
|
|
missense mutation
|
different amino acid
|
|
|
pseudogene
|
untranscribed gene
|
|
|
autopolyploidy
|
fusion of unreduced gametes of the same species
AA --> AAAA |
|
|
allopolyploidy
|
union of gametes from different species
--> aa+AA --> aaAA HYBRIDIZATION |
|
|
production of seedless fruits
|
diploid + tetraploid (colchicine) --> infertile triploid
|
|
|
modern bread wheat
|
allohexaploid
|
|
|
amorphous allele
|
loss of function
|
|
|
hypo morph allele
|
reduction of function
|
|
|
hyper morph allele
|
increase of normal function
|
|
|
neo morph allele
|
gain of novel function
|
|
|
anti morph allele
|
antagonism of normal function (dominant negative)
|
|
|
crossing over in pericentric inversions
|
deletion of some genes
|
|
|
reciprocal translocation
|
no loss (balanced)but alters gene linkage
breakpoint in gene --> mutation |
|
|
robertsonian translocation
|
fusion btw 14 and 21
21 is mostly translocated to 14 --> inherited down syndrome |
|
|
somatic translocation
|
can lead to cancer (leukemia)
|
|
|
ames test
|
test for mutagenicity
auxotrophs on selective medium,add mutagen grow --> substance mutagenic due to spontaneous reversion |
|
|
site specific mutagenesis
|
mutant alleles synthesized and transfered into cell culture or animal
--> way to study mutations of human genes on mice |
|
|
nuclear envelope
|
inner and outer membrane of nucleus
|
|
|
nucleus
|
connected to ER
|
|
|
nuclear lamins
|
build nuclear lamina
deficiency--> muscle dystrophy form intermediate filaments |
|
|
nuclear pore complex
|
NPC
regulate exit of mRNA and entry/exit of proteins |
|
|
nuclear lamina
|
attachement site for chromatin
|
|
|
nucleoplasm
|
chromatin/chromosome containing region
|
|
|
nucleolus
|
building site for ribosome
|
|
|
chromatin
|
protein/ DNA (2:1)
form of interphase chromosomes --> solenoid condensed in metaphase --> silent |
|
|
Histones
|
positively charged
bind to DNA build nucleosomes |
|
|
nucleoplasmin
|
chaperone for assembling histones and DNA
|
|
|
Histone acetylation
|
reversible modification of histone lysine ( N termini)
--> reduced binding to DNA --> destabilization of chromatin |
|
|
Histone methylation
|
-->chromatin highly compacted
|
|
|
Histone phosphorylation
|
Phosphorylation of serines --> formation of metaphase chromosomes
|
|
|
Linker DNA
|
transcribed regions in between nucleosome
|
|
|
telocentric
|
centromere at one end
|
|
|
acrocentric
|
centromere close to one end
|
|
|
metacemtric
|
centromere in the middle
|
|
|
telomeres
|
highly repetetive
3' overhang g rich --> guanine quartets may be capped by proteins AGGGTT in humans |
|
|
catabolite activator protein
|
transcriptional activator
dimer binds to cAMP--> turns lac gene on in absense of glucose by binding to major groove bends DNA--> RNA poly can bind more easily |
|
|
lactose present, glucose absent
|
cAMP-CAP binds--> enhanced transcription
repressor binds inducer (lactose) --> doesnt bind to operator --> TRANSCRIPTION |
|
|
lactose present, glucose present
|
cAMP doesnt bind --> no enhancement
repressor binds inducer (lactose) --> doesnt bind to operator --> very little transcription |
|
|
inducer
|
substrate causing transcription
|
|
|
corepressor
|
substrate causing repression
|
|
|
Trp-operon
|
5 genes, activate each other
end product --> repression attenuation control --> only in prokaryotes |
|
|
attenuation control
|
2-3 --> termination
3-4 --> starvation |
|
|
prophase
|
chromosomes condense
spindle apparatus forms nuclear envelope breaks down |
|
|
metaphase
|
chromosomes line up at equator of cell
|
|
|
anaphase
|
sister chromatids seperate
|
|
|
telophase
|
new nuclear envelope forms
chromosomes decondense |
|
|
meiosis
|
--> cells that are genetically different
reduction division and equational division forms haploid cells |
|
|
sertoli cells
|
nourish developing sperm
secrete seminal fluid (5%) blood testis barrier |
|
|
spermatogenesis
|
spermatogonia (stem cells)
--> primary spermatocytes (4n)--> meiosis1--> secondary spermatocyte (2n)--> meiosis2-->spermatids (1n) --> spermiogenesis -->spermatozoa(1n) |
|
|
hormones in spermatogenesis
|
GnRH (hypothalamus)-->LH/FSH (pituitary)
-->androgens (leydig) FSH --> sertoli |
|
|
nondisjunction
|
error in anaphase 1
|
|
|
nondisjunction of sex chromosomes
|
klinefelters xxy
xyy turners x0 xxx trisomy x |
|
|
polynucleotide kinase
|
labelling by adding ph to 5' end of polynucleotide
|
|
|
blunt end
|
no overlap, always fit together, but can flip
|
|
|
sticky end
|
overlap, needs complementary sequence, melts easier
|
|
|
transduction
|
phages invade bacteria,
can change DNA, may carry DNA from one bacterim to another |
|
|
phage early genes
|
expressed immediately,
code for replication of nucleic acids, in solution--> can clear whole medium, on colonies--> plaque, due to infection of neighbouriung cells |
|
|
ds DNA phages
|
icosahedral,
T even/ T odd, change from linear to circular --> rolling circle --> long DNA mlecule, that is cut and packaged |
|
|
ss DNA phages
|
icosahedral ( x174) or filamentous (M13),
ss converted to ds replicative form, - strand for protein production |
|
|
ssRNA phages
|
filamentous,
f2, MS2 |
|
|
ds RNA phages
|
f6
|
|
|
myoviridae
|
ds DNA phage with contractile tail,
T4, Mu, P1 |
|
|
T4 phage
|
T even,
ds DNA, myoviridae, lytic, icosahedral, gene cluster, rolling circle |
|
|
Mu phage
|
ds DNA,
myoviridae, temperate, integrates into genome with transition mechanism similar to homologous recombination |
|
|
p1 phage
|
ds DNA,
myoviridae, temperate, exists as plasmid in bacterium --> NOT INTEGRATING, icosahedral, encodes cre recombinase (site specific) and lox P site |
|
|
siphoviridae
|
ds DNA with long flexible tails,
lambda |
|
|
microviridae
|
ssDNA with icosahedral heads,
x174 |
|
|
inoviridae
|
ssDNA filamentous,
M13 |
|
|
leviviridae
|
+ ss RNA with tailless icosahedral heads,
MS2 |
|
|
cystoviridae
|
ds RNA with segmented genome,
f6 |
|
|
x 174
|
circular ssDNA,
11 proteins by overlapping genes (3 ORF), icosahedral |
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M 13
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filamentous ss DNA,
circular, attachement to pili (--> only f+ e coli) no lysing, but reduced growth, no packaging, circularizing, replication via rolling circle, ds DNA intermediate |
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filamentous phages
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variable capsid size,
ss DNA circular, big in biotech --> plasmid vectors, T 7 expression system, phagemids, phage display |
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T 7 expression system
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T 7 RNA poly highly specific --> 2 phage promotors in 2 directions--> 2 different RNAs produced with 2 different polymerases
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phagemid
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plasmid that contains 2 different origins ( 1 for ds replication, 1 for packaging),
used in combination with M13 |
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phage display
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gene for protein of interest is integrated into coat protein gene --> 'display' of protein on the outside
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podoviridae
|
stubby tails
T7 |
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MS2
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ss RNA,
icosahedral, attachemnt to pili (only in f + e. coli), 3 proteins only, release by mechanical damage, |
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infection by phages
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--> changes DNA visibly,
1. absorption, 2. eclipse phase, 3. viral replication, 4. maturation, 5. release and re- infection |
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eclipse phase
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seperation of DNA from coat by injection into host,
phage not infectious in this phase, bcs. no infectious particles, landing--> pinning--> tail contraction and penetration--> DNA injection |
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absorption (phages)
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receptor specific --> bacteria can become resistant,
only initially reversible |
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phage replication
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headful length --> each time a bit of genome is cut additionally --> some phages are not infectious --> plaque formation
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maturation ( phage)
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assembly with help of template or scaffold,
T4 self assembly |
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phage recombination
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crossing phages due by coinfecting bacteria,
results in different plaque morphology |
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3 stages of phages
|
1. extracellular,
2. vegetative (virulent), 3. prophage (temperate) |
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lysogenic cycle
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most integrate into genome, p1 in plasmid
some stay latent, like herpes simplex, only some go into lytic cycle --> plaque turbid, once infected --> resistant against other phages of the same system |
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phage conversion
|
phage integrates and alters the phenotype, zB virulence,
in C. diphteria |
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specialized transduction
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integrated phage excises imprecisely --> adjacent host genes are cut out additionally -->
restricted set of bacterial genes transferred into another bacterium, in lambda phage |
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generalized transduction
|
host DNA accidentally packed into phage,
recombines eventually in next host, by chance, |
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RFLPs
|
restriction fragment length polymorphism,
insertion or deletion at restriction site |
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VNTRs
|
variable number tandem repeats,
minisatellites, 5-10 bp |
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SNPs
|
single nucleotide polymorphism,
alterations in DNA involving a single bp, about every 1000 bp, SNP poor and rich regions in genome, source of variation between humans, markers of mutations |
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locations of SNPs
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when in gene --> alter protein,
in regulatory region --> affects protein amount, not in gene vicinity--> genetic markers for locating pathological genes, |
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forward mutation
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wild type to mutant
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reverse mutation
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mutant to wild type
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nucleoplasmin
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chaperone for histone assembly
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rubulavirus
|
mumps
ss RNA paramyxoviridae |
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core DNA
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DNA around nucleosomes
|
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RNPs
|
ribonucleoproteins
--> ribosome and telomerase |
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number of clones required for human genome
|
lambda --> 150000
cosmid --> 75000 BAC --> 15000 YAC --> 1500 |
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partial digestion
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cut genome with less common restriction sites
--> overlap --> you know the ends |
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hybridization
|
to find/ mark/ investigate DNA
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neoplasia
|
tumor
|
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oncogene initial identification
|
retroviruses transduced them to other cells and caused cancer
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colon cancer development
|
1. Loss of APC (ts)
2. Activation of K-ras (og) 3. Loss of DCC (ts) 4. Loss of p 53 |
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Harald zu Hausen
|
HPV
|
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gene thearpy at endothelium
|
clotting factor for haemophilia
can form capillaries to secrete gene product into bloodstream |
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gene therapy for skin
|
skin grafts deliver therapeutic proteins
can be grown from small pieces of skin |
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gene therapy for muscle tissue
|
uptake very quickly but not efficient
duchenne muscle dystrophy |
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gene therapy for liver
|
familiar hypercholesteremia
multiple functions of tissue tissue regenerates |
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gene therapy for lung tissue
|
cells easily accessible
no retroviral vector bcs danger of cancer cystic fibrosis (aerolsole spray) |
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gene therapy for nerv tissue
|
fibroblast can produce neurotransmitters
nerve cells dont divide herpes simplex vector |
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most complicated sequence in humangene therapy
|
regulatory regions
|
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suicide gene therapy
|
causes cell to kill itself
makes cancer cells more vulnerable to chemotherapy 2 ways --> gene directed enzyme production therapy --> virus directed enzyme prodrug therapy |
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gene directed enzyme producing therapy
|
gene taken from tumor and modified
--> injection into tumor where it forms prodrug --> activated only in tumor cells --> harmless to normal cells |
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gene directed enzyme producing therapy
|
virus used as carrier to deliver modified enzymes to cancer cells
no integrating virus --> herpes or cold |
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cancer vaccines
|
in already existing cancers or high risk individuals
--> seperate proteins from cancer cell and immunize against them several in development, only 2 approved (kidney and prostate) |
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SCID
|
severe combined immunodeficiency
genetical absence of functional T- lymphocytes --> B cells not activated properly or also affected treatment--> bone marrow transplant or gene therapy |
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ADA
|
accounts for 15 % of all SCID cases
Ashanti Da Silva and Andrew gobea healed with gene therapy ( retroviral vector) |
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Jesse Gelsinger
|
suffered from OTC deficiency ( urea cycle)
died due to miscalculation of adenoviral amount (-->severe immunereaction) |
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Gendicine
|
adenoviral vector to deliver p 53 tumor suppressor gene
--> used for squamous cell carcinoma (skin cancer) germline mutation of p53 --> retroviral vector!!! |
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non viral vectors in gene therapy
|
Liposome
Cationic polymers naked DNA peptide mediated gene delivery |
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Viral RNA vectors in gene therapy
|
murine leukemia virus
HIV human T cell lymphotropic virus retroviruses. only effective in dividing cells |
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Viral DNA vectors in gene therapy
|
Adenovirus
Adeno associated virus Herpes simplex virus Pox virus |
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|
Adeno associated virus in gene therapy
|
integrating
broad cell tropism potential of target integration low immunogenicity (although already encountered in childhood --> B19 (parvoviridae) fifth disease nonpathogenic infects dividing and non dividing cells requires helper virus |
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Herpes virus in gene therapy
|
high efficiency of transduction
high insertion capacity --> neuronal cells tissue tropism possibly toxic risk of recombination |
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|
vaccinia virus
|
from pox
|
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sindbis virus
|
from toga
|
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foamy virus
|
from retrovirus
|
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onyx virus
|
limited replicating adenovirus
replicates mainly in tumor cells |
|
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gutless vector
|
needs helper virus (helper cell line)
|
|
|
major problem to solve concerning vectors in gene therapy
|
target to proper organsor cell types
|
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gene therapy strategies
|
metabolic manipulation
manipulation of the protein modification of the genome |
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|
somatic cell gene therapy
|
ex vivo--> cells taken out, modified and incubated, returned
in situ --> vector in affected tissue in vivo--> vector into blood stream |
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genes for cancer therapy
|
drug delivery genes
drug resistant genes suicide genes |
|
|
src
|
proto oncogene similar to rous sarcoma
role in cell growth mutation involved in colon cancer progression |
|
|
RFLPs
|
marker in gene sequencing
insertion or deletion in restriction sites --> restriction nuclease doesn't cut detectable with southern blot applied in forensics, paternity tests, detection of genetically modified food |
|
|
genetic markers
|
have to be polymorphic, single locus, neutral
should be on different chromosomes (independent) can also be genes but no ideal choice |
|
|
microsatellites
|
short tandem repeats
highly polymorphic (fast evolving due to slippage of DNA poly over tandem repeats) |
|
|
paternity test with microsatellites
|
1. extract DNA from mother, father, child
2. synthesize 2 oligonucleotide primers each microsatellite 3. amplify microsatellites 4. DNA sequencer --> 3 different microsatellites multiplexed --> to get a relatively secure result --> test 6-12 alleles --> already very small starting material is enough (PCR) |
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|
DNA methylation in eukaryotes
|
1.addition of methyl group to c or a in embryonic stem cells --> changes gene expression
--> can silence viral retrotransposons 2.in somatic cellsat CpG islands (C5) --> transcription factors dont bind --> involved in cancer development as tumorsuppressorgenes are methylated in carcinogenesis |
|
|
e coli hsd methylase
|
methylates a and c
|
|
|
discoveries linked to phages
|
site specific recombination, ssDNA genome, mRNA, DNA ligase, restriction modification, gene therapy, genetic recombination, overlapping genes
|
|
|
jumping genes link to schizophrenia
|
transposon L1 level is elevated in schizophrenics
|
|
|
HIV healed case
|
Berlin man,
can be infected again due to left ver virions integrating virus can stay in stem cells (quiescent) |
|
|
leukemia
|
myeloid or lympheoid
|
|
|
screening cDNA library
|
use expression vector --> mRNA produced to membrane -->binds to radioactive labelled probe
uses: quantify amount of mRNA synthesized from a gene, isolate and sequence a gene for a protein, identify homologous sequences in other organisms |
|
|
s1 nuclease
|
cuts single stranded DNA
--> blunt ends |
|
|
southern blot smear
|
genomic DNA
|
|
|
southern blot fragments
|
cDNA
|
|
|
enveloped viruses
|
Pox, Herpes, Hepadna, Retro, Corona, FLavi/Toga and all - sense RNA except for Reo (rota)
|
|
|
growth factors
|
bind growth factor receptors --> Ras/Raf/Erk -->phosphorylation of jun and fos --> synthesis of D cyclin--> positive control of restriction point
|
|
|
cyclin degradation
|
ubiquitin pathway --> proteasome
|
|
|
no cyclin at all
|
between M and G0
|
|
|
oncogenes
|
genetically dominant
|
|
|
bcl-2
|
apoptosis signalling
|
|
|
angiogenesis of tumors
|
associated with malignancy
|
|
|
Hepatitis A
|
smear infection
vaccine available picorna virus not cancerous |
|
|
Hepatitis B
|
chronic liver infection
cancer associated integration not clear encodes hbx that interferes with p53 |
|
|
Hepatitis C
|
flaviviridae
cancer associated |
|
|
Hepatitis E
|
ss + RNA --> caliciviridae
|
|
|
hepatitis G
|
flaviviridae
|
|
|
transformation of bacteria
|
discovered by griffith
b. subtilis--> formation of intermediate triple strand |
|
|
promiscious plasmid
|
transfer to other species
|
|
|
tra functions
|
cell contact, nicking
must be provided by heelper plasmid in mobilizable plasmid |
|
|
IS elements
|
very simple transposon, genes only for mobilization and insertion
ends show inverted terminal repeats inserts andomly produces staggered cut --> DNA is changed permanently |
|
|
viral envelope
|
left at membrane
|
|
|
diseases asayed wih RFLP
|
sickle cell anemia, phenylketonurea
|
|
|
penetrance
|
rpoportion of people with a particular genetic change who excibit signs and symptoms of a genetic disorder
|
|
|
incomplete penetrance
|
some people with a specific mutation dont develo symptoms (BRCA 1 and 2)
|
|
|
variable expressivity
|
range of signs and symptomsthat can occur in different people with the same genetic conditions
|
|
|
sigma factor
|
subunit of RNA polymerase
|
|
|
mimivirus
|
infects amoeba
|
|
|
metastasis
|
pass blood barrier ( tunica intima - media, -adventitia)
|
|
|
cyclin D and cancer
|
mediates miRNA splicing
mi RNA is increased in breast cancer patients |
|
|
cancer stem cells
|
looses differentiation --> gains proliferation
|
|
|
totipotent
|
3 germ layers + extra embryonic tissue
|
|
|
pluripotent esc
|
3 germ layers
|
|
|
pluripotent adult
|
limited cell types
|
|
|
oligopotent
|
few cell types ( myeloid/ lymphoid)
|
|
|
Denisova and Melanesians
|
share 4-6 %
|
|
|
Eurasians and Neanderthals
|
share 1-4 %
|
|
|
Denisova and Neanderthals
|
genetically different but share anchestor
Denisova bred with other species |
|
|
CRISPR
|
multiple short repeats (sense+ antisense + spacer)
spacerfrom virus --> memory of past exposures forms prokaryotic immune system with cas works analogous to RNAi |
|
|
enhancer and silencer
|
bind transcription factors to fold DNA different
|
