chapters 13, 14, 15 Flashcards
evidence that DNA genetic material
1928 frederick griffith was developing a vaccine for pneumonia, he was looking at two strains a pathogenic strain and a nonpathogenic strain. he mixed dead pathogenic bacteria with living nonpathogenic bacteria and injected mice with this mixture. the mice died when they should have lived- a transformation had occurred- the living bacteria had become pathogenic, or had inherited that trait (griffith didn’t know why)
hershey and chase experiment- used bacteriophages to see if protein or DNA carried genetic material. E.coli was infected by either viruses that had radioactive suffer (to tag protein) or in another group, radioactive phosphorous (to tag DNA) the results were that DNA had entered the cell (phosphorous) and the protein (sulphur) had not, therefor DNA must carry the genetic info
Erwin chargaff-1950 examined base composition of different species. all species had different base compositions relative to each other, but members of the same species had the same number of bases, this made DNA a more credible candidate for the molecule that carried genes. his two rules consist of 1. base pairs vary between species. 2. the number of A’s equals the number of T’s and same for C and G in a species.
DNA structure (product of replication ((conservative stuff)) who discovered DNA
watson and crick looked at ROSALIND FRANKLINS x-ray crystallography of DNA. watson and crick figured out the structure ( watson was familiar with structure of helix in crystallography) model: two strands, double helix, sugar-phosphate backbone with negative charge, bases a-t c-g complementary, two strands antiparallel, dan makes turn (two loops) every 10 bases. A, G purines two rings, C T pyrimidines one ring
a two h bonds t. c three h bonds g.
each DNA strand is template for replication- watson/ cricks second hypothesis: DNA separates and two strands are template for bases which makes two identical strands.- nucleotides line up along template according to base pair rules
semiconservative (what watson and crick predicted, also right); two strands of parental DNA separate and are templates for new DNA Strand- stay with new strand.
conservative-parental strands join back together after- conserve original
disruptive- each daughter is like a strand of mix of original and copied DNA
DNA replication (start)
origin of replication- where replication starts (helicase starts) it is a short stretch of DNA that has specific nucleotide sequence , proteins recognize this sequence and attache to DNA - open DNA makes a replication bubble. -replication fork- y shaped region where DNA strands are being separated - helicase- untwist and separate strands of DNA at replication fork -single stranded binding proteins- binds to opened DNA strand so they don’t re attach before nucleotides added ( a and t attracted to each other like c and g of complementary strand) -topoisomerase- relieve stress in strand by untangling the DNA ahead of the replication fork.
- E.coli- single origin of replication( all bacteria) eukaryote- multiple origins of replication that join together as DNA is synthesized.
- enzyme that makes DNA ( DNA POL) cannot start synthesis- in can only add nucleotides (3) -primer- is required to start chain, it is a short strand of RNA, it is 5 to 10 nucleotides long -primase- adds and makes primers for DNA strands
DNA Replication ( middle/ end)
DNA pol 3 adds nucleotides after primer is set, DNA pol 1 replaces primer with DNA nucleotides. in eukaryotes nucleotides are added 50 per second and 500 per second in bacteria.
-nucleotide- base, sugar and THREE PHOSPHATE groups until it is added to strand, the two phosphate molecules break off from third, this reaction is exergonic and fuels polymerization- energy coupling
DNA elongates antiparallel to parent template strand because DNA strands have directionality. DNA POL CAN ONLY ADD TO 3’ END OF PRIMER AND NUCLEOTIDEs so DNA ELONGATES IN 5’ TO 3’ DIRECTION. (template 3- to 5’) DNA pol adds as fork processes.
leading strand- moving in 5’ to 3’ direction and leads synthesis of DNA after origin of replication
lagging strand- strand going towards origin of replication, DNA must still be synthesized in 5’ to 3’ direction so primase waits for DNA fork to open, adds primer, DNA pol 3 adds nucleotides in 5’ to 3’ to the last primer.
okazaki fragment- fragment of lagging strand ( 1000 - 2000 pairs of nucleotides in E.COLI)
DNA ligase- forms bond between DNA fragments after primer is replaced
DNA replication complex
many proteins involved with DNA replication, DNA moves through complex. the complex acts like groups of factories anchored to the nuclear matrix and DNA pol reels DNA through, the lagging strands are looped back through complex
proof reading/ repairing DNA
mistakes occur 1 in 10 exponent5 but complete DNA molecule has 1 in 10 exponent10 mistakes.
DNA pol proof reads DNA nucleotides and removes mistakes -mismatch pair- evade proof reading or enzymes remove or replace incorrectly paired nucleotides from replication errors but errors can also occur outside of replication.- mutagens can cause changes in DNA and therefore changes in phenotype (mutation).
- there are 100 repair enzymes in E.COLI and 130 in humans.
mechanism of repair- nucleotide excision repair-takes advantage of base pairs NUCLEASE- enzyme that cuts out damaged DNA- gap filled by DNA pol with new nucleotides and fixed together by DNA ligase
ex: SKIN CELLS - thymine dimers occur (two thymines connected) this occurs in people who have xeroderma pigmentosum disorder- where their repair enzymes are inactive which makes them more likely to acquire cancer so they are hyper sensitive to sunlight.
evolution
permanent change in DNA is a mutations, mutations change the phenotype. change can be good bad or neutral. if a mutation occurs in germ cells it can be passed on through generations. mutations are the source of variation of alleles in a population and natural selection determines traits of a new species
end of DNA
chain of DNA is linear and replication machinery can only complete 3’ so the end is shorter ( uneven) so there are TELOMERES at the ends which are a buffer zone that post ones degradation of the DNA molecule
human telomeres are ttaaggg and they are shorter in cells that divide a lot they may also have a role in the aging process
- telomerase catalyzes the lengthening of telomeres in GERM CELLS to ensure genes are not lost from generation to generation
- telomerase not active in cancer cells- make cancer life longer
chromosomes
carry genetic info, in bacteria there is a single chromosome, and only a region where the DNA resides: the nucleiod. in humans one single strand of DNA makes up a chromosome. chromatin is made of DNA and proteins that make up a chromosome- during the cell cycle the compactness of the chromatin changes greatly.
interphase- extended chromatin -mitosis-chromatin condenses- metaphase*chromosome most distinguished
heterochromatin- interphase visible chromatin (AT CENTROMERE) euchromatin- less compact
- variation in cell density of chromatin necessary for different cell processes- meiosis, mitosis and gene activity
- histone modification has an impact on density of chromatin
chromatin packing
DNA- 2nm across HISTONES- proteins in first level of DNA packing ( 100 amino acids and one fifth of them are positive- attracted to negative backbone of DNA)-H2A, H2B, H3 H4- during DNA replication/ transcription histones briefly no one knows where they go
NUCLEOSOME- 10 nm (beads on string)DNA wrapped once around 4 nucleotides- nucleosome made of two sets of four so 8 and DNA wrapped around it twice. DNA between nucleosomes called linker DNA histones tail extends out from nucleosomes
FIBER- 30nm H1 histones involved (special) nucleosomes coil.
LOOPED DOMAINS 300 nm ( 30nm fibers form loops) topoisomerase also used
METAPHASE CHROMOSOME- 1400nm looped domains coil and form compact chromosomes, one chromatid 700nm***specific genes always end up at the same place. this indicates that packing steps are highly precise and specific.
genetic engineering+ DNA cloning
-direct manipulation of genes for practical purposes
DNA CLONING- gene cloning- genes small segments of DNA the rest is noncoding DNA and distinctions between gene and noncoding DNA subtle.-scientists work with specific genes and develop methods of preparing well defined segments of DNA by using bacteria (E.coli). BACTERIA have plasmids which are small circular molecules of DNA that replicate separately from bacterial chromosome which may be from another bacterium and used when necessary (changing environment) scientists take plasmids and genetically engineer it - - like adding human genes- now RECOMBINANT DNA - gene from 2 different sources.made in vitro (test tube)- plasmid returned to bacterium, recombinant bacterium make many copies of gene or protein that gene codes for. MEDICAL USE- proteins / hormone production (insulin)
restriction enzymes
cut DNA nucleotides at specific spots - this protects bacteria from foreign DNA (Virus) can be used to make recombinant enzymes
restriction site- short sequence of DNA nucleotides that restriction enzyme recognizes and cuts out specific DNA- methyl groups attach to adenines and cytosines so they aren’t cut out by restriction enzyme.
most restriction sites are symmetrical
restriction fragments- pieces of DNA cut out
*** all copies of particular DNA molecule always yield same fragments when exposed to some enzyme
GEL ELECTROPHORESIS- separate picture of nucleic acid fragments by length- can see who’s DNA is who’s, expose same to restriction fragments, certain parts cut at specific locations, if another sample is cut and looks identical the two samples must contain the same DNA
nutritional mutations in neurospora
beadle and tatum worked with bread told and disabled the genes one by one looking for changes in the phenotype
experiment: neurospora haploid organisms wild type has simple nutritional requirements (inorganic salts, glucose, biotin). cells individually plated. individual cells placed on dish with complete growth medium then x rayed to induce mutations. surviving cells formed new colonies. then tested for ability to grow on minimal medium- identifying them as nutritional mutants. mutant cells from each colony placed in a series of vials with minimal medium and one additional nutrient. the supplement that allowed growth indicated defect from induced mutation (different colonies blocked at different steps in metabolic pathway- either lacked enzyme or enzyme was changed)
supported 1 gene = 1 protein hypothesis
products of gene expression
many proteins constructed from two or more polypeptides each polypeptide specified by gene. eukaryotic genes code for multiple closely related polypeptides— alternative splicing— some genes code for functional RNA that are not transcribed (tRNA, rRNA) therefore 1 gene does not = 1 protein
genetic code
4 bases. 20 aa. codon- 3 nucleotides that code for specific amino acid. eukaryote codons are non overlapping.-template strand- strand of DNA used for mRNA synthesis, same strand always used for certain gene. RNA assembled using base pair rules (u for t) RNA made antiparallel to DNA strand- codons written 5’ to 3’ and also read that way too.
61 different codes for amino acids, aug start codon other two start and stop codons. hug codes for methionine- all RNA chains start with met
transcription
RNA polymerase opens DNA and adds nucleotides, it doesn’t need a primer and adds them in the 5’ to 3’ direction.-promoter- sequence where RNA pol starts(farthest upstream) -terminator-signal to end sequence. -transcription unit- stretch of DNA transcribed into RNA. -
INITIATION- promotor occurs upstream before start point of transcription RNA pol 2 binds to promotor - transcription factors- determine binding site of RNA pol 2 and initiation of transcript - factors bind to promoter first then RNA pol2 binds -transcription initiation complex- transcription factors, promoter, RNA pol 2. TATA box- promoter sequence.
ELONGATION- RNA pol moves along DNA and opens up 10-20 nucleotides at a time and adds nucleotides to 3’ and of DNA (5’ end of RNA) - RNA peels off of DNA template and DNA closes up behind it. the length of RNA reflects how long template was, many polymerases simultaneously transcribe gene
TERMINATION (bacteria terminator sequence of DNA- RNA POL detaches detaches and releases transcript) EUKARYOTES- polyadenlylation- the signal for it is aauaaa and it adds poly a tail to 3’ tail- it is used for protection against degradation as well as transcription termination, export from nucleus and translation. 5’ cap is added to the 5’ end is used for protection against degradation as well as helps the RNA bind to ribosomes
UTR untranslated region, before and after start and stop codons
split genes and RNA splicing
EUKARYOTES** removal of RNA portions called RNA splicing. average pre mRNA is 27000 nucleotides and it is reduced to 1200 nucleotides DNA coding for poly peptide is not continuous INTRONS-noncoding segments EXONS coding and expressed through translation.- except UTR-not translated but also not cut out. PRE mRNA- has introns and exons -mRNA intones have all been cut out axons are joined together to form a continuous coding sequence alternative RNA SPLICING *single gene encode more than one type of polypeptide depending on which segments of DNA are treated as introns and exons therefore one gene codes for many types of proteins.
-spliceosome- complex made of protein and RNA segments bind to nucleotides on an intron, the intron is cut and released spliceosome also joins exons- small RNA in spliceosome catalyze the process.
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