Chromatin and Chromosomes Flashcards
Nucleoid
is an irregularly shaped region, occupying 1/3 of a prokaryotic cell, where its genetic material is localized
Lysozyme
is an enzyme able to lyse the bond between peptidoglycans in the bacterial wall and can be found in human epithelial secretions
lysozyme is more effective on what kind of bacteria
gram +
what’s the difference between gram + and gram- bacteria
Gram + has a much thicker peptidoglycan layer than gram -. Gram - also has an external membrane on top of the peptidoglycan
how do we isolate the nucleoid?
~Treat the E.coli cells with lysozyme
~induce osmotic shock
~low speed centrifugation
what kind of genes does Plasmidic DNA code for
for non-essential traits, that could contribute to an evolutionary advantage.
~it has 1000’s of bases
~circular DNA
what kind of genes does Genomic DNA code for
essential informations, house-keeping genes
~has 1000000’s of bases
~circular DNA
vector
piece of DNA which can replicated autonomously and can accept a piece of DNA from another source
why do plasmids make good vectors?
~They are small and easy to manipulate
~There are many within a cell and so many copies are available.
~They always contain a site of origin of replication and almost always contain a gene giving resistance to a specific antibiotic - which allows for selection in lab situations.
~Can contain a marker which allows for selection and have a poly linker or MCS
Bacterial conjugation
method by which bacteria exchange info, through a sex pillus
how is DNA organized in a bacterial cell?
it is supercoiled and attached to a protein core
what effect does DNase have on bacterial DNA
it creates small nicks in the super coils
each loop in a E.coli genome corresponds to a
structural domain
why is bacterial DNA organized in domains?
because the E.coli cell cannot contain a completely relaxed chromosome and only relaxed DNA can be transcribed
Chromatin
was coined by alexander flemming in 1882
it is stained by the Feulgen reagent (red)
what is chromatin made of?
~DNA
~Histones
~Non histonic proteins (neutral/acidic)
~RNA
costitutive heterochromatin
always in compact form, is usually repetitive DNA
facultative heterochromatin
is present depending on cell type
how long is the human genome
2m
the nucleus diameter is
5-10 micrometers
chromosome length is
1.6-8.2 cm
how to study proteins associated with DNA
~treat with DNase
~use salts to break interaction between histones and bases of DNA
~use polyacrylamide gel to separate the proteins
~treat with SDS - makes the proteins negatively charged
~heat and use betamercaptoethanol to denature (it breaks disulfide bridges)
electron dense beads on the beads in a string structure are made of?
DNA and histone proteins
the beads on a string structure is
a left handed solenoid supercoiling
beads are
histone octamer around which 1.65 left handed turns of DNA are wrapped.
string is
linker DNA ranging from 8bp to 114 bp
nucleosome
is a core of 8 histone proteins and linker DNA wrapped around it
the tails of a protein are called the - terminal
N terminal
histones are
small polypeptides
~positively charged
~enriched in basic aa: Arg/Lys
~pL=9~10
which histone has a more conserved sequence? H4 or H1
H1
the minor groove faces away/towards the histone?
towards. Note that there are mostly AT pairs here
What is the 2nd level of condensation?
the beads on a string structure coils into a 30nm diameter helical structure
~this is not present along the whole chromosome
explain the solenoid model of condensed chromatin
the DNA does not cross the superhelix longitudinal axis, the linker DNA is less accessable
explain the zig-zag model of condensed chromatin
the linker DNA is longer and more accessible
what is the 3rd level of condensation
loops of 300nm fibers and nuclear scaffold
what is the 4 level of condensation
the loops form rosettes (6 loops per flower) which then form coils made of 30 rosettes
each chromatid is made of - coils
10
what modification of histone tails are possible?
acetylation, methylation, ubiquitination, sumolyation and phosphorylation.
acetylation
Usually occurs on lysine. The specific enzyme is Histone Acetyl Tranferases (HAT) they acetylate lysine. The reverse is done by Histone Deacetylase (HDAC)
Acetylation changes the properties of lysine (which are basic residues and so have a positive charge) by removing the positive charge. This change in charge makes it more difficult for the histone to interact with DNA, and so it favours the opening of the chromatin structure.
Explain methylation
Lysine can also be methylated. HOWEVER, it cannot be both acetylated and methylated. they are competing reactions and are mutually exclusive. It is possible for lysine to be di and tri- methylated also. Methylation does not remove the positive charge and so does not favor the opening of the chromatin.
Ubiquitination
It is a type of acylation which uses a small polypeptide of 7-8 amino acids called ubiquitin. It also acts on lysine.
It occurs through a slightly complex reaction. It requires 3 different enzymes; E1 (which is ubiquitinated through ATP hydrolysis) the ubiquitin is transferred to E2 which uses E3 (an enzyme which can recognise both the target molecule and the E2 enzyme carrying the ubiquitin
phosphorylation
May either activate or inactive a protein. It requires an alcohol group to be present in the protein. This is present in serine, threonine and tyrosine. A phosphate group is added, requiring ATP and catalysed by kinases. Phosphatases reverse the reaction
