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Carden Flashcards

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1
Q

What do light microscope images depend?

A

Optical setup (of microscope)
Light Alteration by specimen
[a] Diminished INTENSITY
[b] Altered WAVELENGTH (or selective change in its range)
[c] Altered PHASE
[d] Altered INTERFERENCE
(or a combination of some/all of these)

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2
Q

How does Fluorescence Microscopy work?

A

1) first barrier filter: lets through only blue light with a wavelength between 450 and 490nm
2) Beam splitting mirror reflects light below 510nm but transmits light above 510nm
3) seconds barrier filter cuts out unwanted fluorescent signals passing the specific green fluorescein emission between 520 and 560nm

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3
Q

In the modern era cell sectioning is achieved with what?

A

Microtomes

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4
Q

How is a specimen generally prepared for light microscopy?

A

Fixed and embedded
Sectioned (cut thinly enough for light to pass through)
Stained
mounted and viewed

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5
Q

How is Specimen fixation achieved for light microscopy?

A

Chemical cross-linking

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6
Q

how is embedding achieved for light microscopy

A

Molten wax, allowed to cool
inflitration with wax requires specimen dehydration (alcohol
but also can use acrylates (plastics) and water-soluble media

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7
Q

How is sectioning achieved in light microscopy?

A

Microtome

Specimen bath to float and flatten sections

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8
Q

How is staining achieved for Microscopy?

A 
Histological dyes 
H&E = Haematoxylin (DNA purple) 
eosin (Protein pink) 
Fluorescent compound 
DAPI (DNA) derivatised antibody (ultraspecific) 
More specific stains
More specifc stains
Osmium (membranes) 
silver (v limited cytoskeletal)
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9
Q

Advantages of light microscopy?

A

The light microscope is particularly useful for looking at the general size and shape of cells.
It provides an overview of organization into tissues (histology) and can visualise larger substructural features (such as the nucleus). It also indicates the general localization of substances in the cell.
Most powerfully, LM provides pictures of processes and phenomena associated with LIVING cells (such as movement, temporal changes, mitosis, etc.).
Images from living cells can be recorded (with video camera attachments) in real time and speeded up or slowed down, image enhanced by computer, etc. to reveal details not readily apparent to the eye.

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10
Q

How are samples prepared for TEM

A

Vacuum: dead specimens only
Special fixatives: glutaraldehyde, osmium
Ultrathin sections: ultramicrotome, diamond knives, acrylate embedding, copper grids
NO CONTRAST

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11
Q

(heavy metal) shadowing

A

Heavy metal evaporated from a filament shadows the specimen
a strengthening film of carbon evaporated from above
The replica is floated onto the surface of a powerful solvent to dissolve away the specimen
The replica is washed and picked up on a copper grid for examination

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12
Q

What isotope is used to label DNA

A

32P (can also be used to label phospho transfer e.g. to proteins by kinases, but also DNA end labelling, etc.)

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13
Q

What isotope is used to label protein

A

35S labels proteins (via Met and Cys)

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14
Q

What is the Hershey Chase experiment?

A

Proved that DNA is the genetic material using the radioisotopes 32P and 35S
Phages (with 32P and 35S) infect sample (Ecoli) and when tested Ecoli only contained 32P showing that DNA is the genetic material

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15
Q

What is the Meselson Stahl experiment (1958)?

A

Proved semiconservative replication using heavy nitrogen N15 and light nitrogen N14

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16
Q

What are the pulse-chase experiments?

A

Cleavage of gp160 and gp120 of HIV
U= uncleaved
C= cleaved

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17
Q

How to perform (micro) Autoradiography

A

Coat specimen with gelatine and silver salt mixture (in the dark)
Reduce the activated Ag+ to Ag metal
wash away soluble Ag+ leaving insoluble Ag metal
Observe with electron microscope

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18
Q

What is Serum

A

the liquid part of clotted blood (minus fibrinogen and other clotting factors)

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19
Q

How are monoclonal antibodies made?

A

Large quantities of a single type of antibody molecule can be obtained by fusing a Bcell (taken from an animal injected with antigen A) with a tumour The resulting hybrid cell divided indefinitely and secretes anti A antibodies of a single (monoclonal) type

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20
Q

Immunoblot ( western blot with antibody detection)

A

the sample undergoes protein denaturation, followed by gel electrophoresis. A synthetic or animal-derived antibody (known as the primary antibody) is created that recognises and binds to a specific target protein

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21
Q

When did Mendel form the basic laws of heredity

A

1800’s

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22
Q

What did Fred Griffith?

A

1928 A transforming principle Streptococcus pneumoniae

using Mice S and R strain

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23
Q

Oswald, Avery and Macleod

A

1944 Transforming principle S strain cell
Fractioning of classes of molecules
DNA carries heritable information

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24
Q

Watson & Crick 1953

Also Chargaff

A

a double-stranded DNA molecule globally has percentage base pair equality: %A = %T and %G = %C

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25
How is DNA organised in prokaryotes?
DNA normally in one circular chromosome located in the nucleoid region Additional, small pieces of DNA can be present (plasmids)
26
How is DNA organised in Eukaryotes?
Special Compartments Most DNA is in the nucleus, organised into linear chromosomes Some DNA also in mitochondria, organised into circular chromosomes Plasmids can be maintained in many eukaryotes
27
How big is the Human Genome?
Haploid amount is= 3 billion bp
28
Chromatin
The complex of DNA and proteins
29
Albrecht Kossel
1853-1927 DNA was observed to be associated with protein | It is this association between DNA and protein that underpins the organisation of DNA molecules in cells.
30
Nucleosome
nucleosome is a basic unit of DNA packaging in eukaryotes, consisting of a segment of DNA wound in sequence around eight[1] histone protein cores.[2] This structure is often compared to thread wrapped around a spool.[3]
31
What are the 4 different subunits in a standard histone?
four different subunits are called H2A, H2B, H3 and H4
32
What is a solenoid structure?
Nucleosomes assemble into the chromatin fibre
33
What do chromatin fibres form
bundles loops are coiled/bundled around scaffold proteins
34
Euchromatin
Some regions contain genes that need to be actively expressed. These regions are packaged less densely and are called Euchromatin
35
Heterochromatin
Other regions do not need to be actively expressed these are packaged densely and are called heterochromatin
36
What 2 groups can Heterochromatin be further divided into?
Constitutive Heterochromatin: contains no genes is always densely packed e.g. telomeres, centromeres, most of the Y chromosome, the ‘extra’ X chromosome in females. Facultative Heterochromatin: Contains genes that are needed only under certain conditions these regions can become less densely packaged when the genes need to be expressed
37
“Karyotyping”
is the identification and cataloguing of the chromosomes of an individual.
38
What convention are DNA sequences always written to
5' to 3'
39
How many replication origins can a Eukaryotic chromosome have
Several
40
how many replication origins do bacterial, viral and plasmid DNA have?
1
41
What direction do DNA polymerases synthesise DNA in ?
5'-3'
42
Why must the template strand be primed? (have a double stranded start)
DNA polymerase can only add nucleotides to a 3' end.
43
why are different types of polymerase needed in organisms?
All organisms contain repertoires of DNA polymerases with slightly differing properties, which have different jobs in the cell. There is always at least one polymerase that has the main copying job during DNA repair, other DNA polymerases may be involved in DNA repair or in maintaining the small genomes of organelles (mitochondria and chloroplasts have their own
44
What kind of reaction does the DNA polymerase run in when its heading away from the origin in the 5' to 3' direction
continuous
45
Some ezymes needed for replication are?
Primase- synthesize short RNA primers on the lagging strand | Topoisomerase- prevents DNA ahead of fork becoming overly coiled
46
What is the role of primase
part of the replication machine | Enzyme synthesize short pieces of RNA that act as primers for synthesis of Okazaki fragments
47
what are the two main sources of point mutations in DNA?
Inaccuracy in DNA replication | Chemical damage to DNA
48
Telomerase
Adds additional telomere repeats to template strand | restores chromosome ends
49
What are Tautomers?
forms of a chemical that contains the same number of atoms but have differenct structures that can spontaneously interconvert (causes mutations)
50
How can it cause a transversion mutation?
If nucleotide adopts the rare tautomeric form during the short time interval it takes for the DNA polymerase to copy this nucleotide
51
DNA polymerases and proofreading?
some have 3'-5' exonuclease activitty
52
What special mechanism exists to ensure that nucleotides are repaired on the newly synthesized strand, rather than the old strand:
Cell attached Methy groups to adenines in the DNA at infrequent intervals Methylation of the new strand lags behind the DNA replication process This gives the repair-machinery time to identify misincorporated nucleotides, and exchange the nucleotide on the daughter strand
53
Slippage during DNA replication (in simple repeat DNA):
The reason for this is that, during synthesis of the new daughter strand by the DNA polymerase, the ends of the newly copied DNA frequently detach from the template strand. In most types of DNA, they only have one way of annealing back to the DNA. The DNA polymerase therefore simply waits until the DNA has re-annealed, then resumes the copying process. In repeat regions however, the newly copied DNA can re-anneal with repeats other than the one it was originally annealed to. This can lead to a repeat being copied twice, or not being copied at all.
54
What is depurination
Loss of base groups from the sugar/phosphate backbone | 5000 bases per cell per day
55
What is deamination
Loss of base groups from the sugar/phosphate backbone occurs naturally: 100 bases per copy of genome per day
56
Thymine dimers?
Two adjacent thymine bases have become covalently attached to each other to form a thymine dimer. Skin cells that are exposed to sunlight are especially susceptible to this type of DNA damage.
57
Nucleotide excision repair
In other cases, it is not clear which nucleotide received the damage. For example, a wrong nucleotide may be incorporated because the template nucleotide was in the wrong tautomeric conformation. If this is not immediately detected, the repair machinery can only see an incorrect base pair, but cannot necessarily detect which of the two bases is the wrong one. In these cases, and excision repair mechanism is often used that excises and re-synthesizes a longer stretch of DNA around the point of damage.
58
Exonucleases
are processive (they remove multiple nucleotides in one go) usually sequence-independent (they will digest any DNA sequence) often directional (they will act in 3’->5’ or 5’-3’ direction, but not both)
59
Endonucleases
``` not processive (they cut once) usually sequence-dependent (cut only at a specific DNA sequence) ```
60
Alkylating agents
Add chemical groups to existing bases in a DNA strand which permanently changes their base pairing pattern
61
Intercalating agents
``` forcing the sugar-phosphate backbone to stretch. Intercalating agents are a special class of chemicals that have a flat shape, which can be inserted easily between the stacked bases in the DNA double helix. This distorts the normal distance between the base pairs, and can lead to insertion mutations. ```
62
Non homologous end joining?
Processing of DNA end by nuclease | end joining by DNA ligase
63
Homologous recombination
Processing of broken ends by special nuclease | Double strand break accurately repaired using undamaged DNA as template
64
Recombination errors cause chromosomal mutations
Chromosomal mutations are large-scale rearrangements in chromosomal DNA. One of the main reasons for their occurrence is inappropriate recombination (ie recombination between sequences that do not belong to sister chromatids, that are not really identical, and that should not therefore recombine under normal conditions.)
65
What are the non coding sections of DNA?
Promoters, terminators, UTRs and introns
66
How are genes in a bacteria organised differently than in a Eukaryote?
bacterial gene consists of a single stretch of uninterrupted nucleotide sequence that encodes the amino acid sequence of a protein (or more than one protein). In contrast, the protein-coding sequences of most eukaryotic genes (exons) are interrupted by noncoding sequences (introns). Promoters for transcription are indicated in green.
67
What is an operon?
. Clusters of genes transcribed as a single mRNA molecule are common in bacteria. Each of these clusters is called an operon because its expression is controlled by a regulatory DNA sequence called the operator (green), situated within the promoter.
68
RNA polymerase
always moves in the 3′-to-5′ direction and the selection of the template strand is determined by the orientation of the promoter

Molecular and Cellular biology (1 decks)

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