Molecules, Genes and Disease Flashcards
What is the function of the mitochondria?
ATP synthesis
What are the functions of the plasma membrane?
Transport of ions and small molecules
Cell morphology and movement
What are the functions of the cytoplasm?
Metabolism of carbohydrates, amino acids and nucleotides
Fatty acid synthesis
What are the functions of the nucleus?
DNA synthesis and repair
RNA synthesis and processing
Ribosome assembly
What are the functions of the Golgi?
Export of proteins
Detox reactions
What is pK
How readily an acid will dissociate, and therefore how strong it is
How can amino acids be classified?
Aliphatic or aromatic
polar, uncharged, non polar, negatively charged or positively charged
What is the isoelectric point?
The pH at which a molecule has no overall charge
Why does haemoglobin have a sigmoidal affinity curve for oxygen?
Because it can exist in a tense or relaxed state. When one oxygen molecule brings it changes from the low affinity T state to the high affinity R state. This makes it more sensitive to slight changes in partial pressure.
What are the functions of the smooth and rough ER?
Protein synthesis and transport
Membrane synthesis
Lipid and steroid synthesis
Detox reactions
Describe the Bohr effect
Presence of protons and carbon dioxide in the blood make it more acidic, reducing the O2 affinity of haemoglobin, causing increased oxygen deposition in the more metabolically active tissues.
What affect does BPG have on haemoglobin and what does BPG stand for?
Reduces O2 affinity, shifts association curve to the right, promotes oxygen deposition in the tissue.
2’3-bisphosphoglycerate
What affect does CO have on haemoglobin?
Binds irreversibly, reducing the amount of haemoglobin available for oxygen transport.
What is the mutation and a.a. Change in sickle cell anaemia?
A to T point mutation
Glutamic acid to valine substitution
What is the consequence of the sickle cell mutation?
Valine is hydrophobic, so creates a hydrophobic ‘sticky’ region in the beta chain, when in T state the haemoglobin polymerises causing RBCs to adopt a sickle shape. Can occlude small vessels.
What factors promote sickle cell crisis?
Anything that reduces O2 concentration e.g. Smoking, obesity.
What are the complications of sickle cell anaemia?
Haemolytic anaemia
Jaundiced due to excess bilirubin due to increased RBC turnover
What are the two types of thalassemia, and why are they different?
Alpha - due to decreased or absent alpha haemoglobin chains, appears before birth.
Beta - due to decreased or absent beta haemoglobin chains, appears after birth because fetal haemoglobin contains alpha and theta chains.
Define Vmax
Maximal rate when all enzyme sites are saturated with substrate
Define Km
Substrate concentration that gives half the maximal rate of reaction
What do the intercepts on the lineweaver-burk plot show?
X axis - negative reciprocal of Km
Y axis - reciprocal of Vmax
What kind of inhibitors bind covalently and destroy enzyme function?
Irreversible
What are the two types of reversible inhibitors?
Competitive -bind to active site, affect kM not Vmax. Can be overcome by increasing substrate concentration.
Non competitive - bind away from active site, affect Vmax not Km. can’t be overcome by increasing substrate concentration.
What is allosteric control?
Multi subunit enzymes which have multiple binding sites, either for the binding of inhibitors or activators, or for one substrate to bind to, promoting the binding of a second substrate.
Give an example of an allosteric enzyme and its control
Phosphofructokinase is activated by AMP and inhibited by ATP
Name two mechanisms of regulation of enzymes and give examples of them - not including allosteric control.
Covalent modification e.g. Phosphorylation
Proteolytic activation - inactive enzyme zymogen has protein cleaved to produce the active form. E.g. Trypsinogen and trypsin.
What is the benefit of enzyme cascades?
Allows amplification of a signal very rapidly
Describe the clotting cascade - 5 points.
Inactive zymogens of tissue factors are present in the blood at low concentrations
Damage to endothelium creates a cascade of activation resulting in a insoluble fibrin clot
Factors with gla domains bind to endothelial cells and allow rapid activation of downstream effector molecules.
Activated thrombin feedback to enhance the conversion of factors V, VIII, and XI to activated forms
Clotting is stopped by the removal of activated proteins, proteolyric digestion and the binding of inhibitor molecules.
What are the purine and pyramiding RNA nucleotide bases?
A and G are purine
C and U are pyramidine
What are the purine and pyramidine DNA nucleotide bases?
A and G purine
C and T pyramidine
In which direction is it conventional to write base sequences?
5 prime to 3 prime
Which enzyme unraveled the DNA helix?
DNA helicase
What enzyme catalysed the condensation reaction between DNA nucleotides?
DNA polymerase
Which direction does the nucleotide chain grow in DNA replication?
5 prime to 3 prime
What is different about the synthesis of the leading and lagging strand?
Leading strand is synthesised continually
Lagging strand is synthesised in Okazaki fragments
What enzyme joins the Okazaki fragments?
DNA ligase
What happens in stage G1 of the cell cycle?
Cell prepares for DNA replication, cellular contents duplicated
What happens during stage s of the cell cycle?
DNA replication
What happens during stage g2 of the cell cycle?
Checks and repairs duplicated DNA
Define gene
A length of DNA which codes for a protein or RNA
Define allele
Specific version of a gene
What are the three main patterns of inheritance?
Autosomal dominant
Autosomal recessive
X linked
Define codominance
When both alleles are expressed in the heterozygote, e.g. AB blood type
Define complementation
When more than one gene is responsible for the phenotype
Define co-inheritance
Genes on the same chromosome tend to be inherited together, they after linked. The closer together their locus the less likely they are to be separated by crossing over.
What are the tree stages of transcription and translation ?
Initiation
Elongation
Termination
Describe the initiation stage of transcription
Promoter region recognition
Binding of transcription factors and RNA polymerase to the TATA box
Describe the process of elongation in transcription
RNA is transcribed, the DNA is read 3 prime to 5 prime
Describe the process of termination in transcriptioN
sequence dependent termination of RNA chain growth
Describe the process initiation in translation
AUG codon recognised and ribosomal sub units bind around it to form a functional ribosome
Describe the process of elongation in translation
tRNA brings amino acids, ribosome catalysts the condensation reaction between them. mRNA is read 5’ to 3’ and amino acids run N to C
Describe the process of termination in translation
Stop codon recognition and dissociation of the ribosome
In what three ways is RNA processed?
Capping of 5’ end
Tailing of 3’ end with poly A tail
Splicing of introns
What is a codon?
Three base pairs that code for an amino acid
Why is DNA code said to be degenerate?
There are more 3 letter combinations than there are amino acids, so there are several ways of coding for each amino acid.
Which type of RNA exits in 1000s of forms with just a few copies of each form?
mRNA
Which type of RNA exists in many types with many copies of each type?
tRNA
Which type of RNA exists in just a few types but there are 1000s of copies of each type?
rRNA
In what 5 ways is protein synthesis different in bacteria?
Simpler promoter region Coupled transcription and translation Shortlived, non processed mRNA 70s ribosomes Different RNA polymerase (can be a target for drugs)
Describe constitutive secretion and give an example of a protein produced this way
It is a continuous process
Proteins are packaged into vesicles and released continuously by exocytosis
E.g. Collagen, serum albumin
Describe regulated secretion and give an example of a protein secreted this way
Protein is packaged into vesicles but only released in response to a signal
E.g. Insulin
Describe the secretory pathway (8 points)
mRNA recognised by the free ribosome
N-terminal signal sequence produced
SRP (signal recognition protein) recognises sequence
SRP directs ribosome to the ER membrane where it attaches to a peptide translocation complex
SRP dissociates
Protein synthesis continues, protein is fed into ER lumen
Signal sequence removed by signal peptidase
Ribosome dissociates and is recycled
What organelle carries out n-linked glycosylation?
ER
What organelle carries out the majority of o-linked glycosylation?
The Golgi
What is proteolytic processing?
Removal of a signal peptide to convert inactive protein to an active one
Describe the formation of insulin
Signal sequence removed from preproinsulin
Disulphides bridges form on proinsulin
C peptide is remove forming insulin
Describe how collagen is made in the ER (3 points)
Protein synthesised and signal peptide cleaved
Protein is modified by hydroxylation and addition of n-linked oligiosaccharides and galactose
Disulphides bridges and triple helix of procollagen form
Describe how collagen is modified by the Golgi
Glucose is added to o-linked galactose
How does collagen go from the Golgi to a fibrin strand?
Secreted in vesicles into extra cellular space
N and c terminal properties are cleaved
Molecules associate laterally into fibrils
Fibrils aggregate to form fibre
Describe gene cloning (3 points)
Plasmid cut using restriction enzymes and gene of interest is added to create a recombinant plasmid
Plasmid is introduced to bacterium
Bacteria multiply
Describe restriction analysis (2 points)
Restriction enzymes are bacterial enzymes which recognise specific DNA sequences know as restriction sites and cut the DNA at that point
This creates sticky ends which can be joined using DNA ligase
Describe DNA sequencing (2 points)
Fluorescent/radioactively stained ddNTPs and dNTPs are added to a mixture containing a DNA template strand with polymerase to create many strands of varying length
The fragments of different sizes can be separated using electrophoresis and the sequence determined
Describe gel electrophoresis
Solution with DNA fragments is placed in well at negative electrode
Negatively charged DNA molecules are attracted to the positive electrode
Larger fragments are slower so do not travel as far
Known DNA lengths are used for reference
How does PCR work and what is it used for?
Uses thermal stable Taq polymerase to amplify DNA fragments 1- denaturation at 94-96 degrees c 2- renaturation at 50-65 degrees c 3- DNA synthesis at 75-80 degrees c This cycle is repeated many times
Describe SDS page
The detergent SDS is adds to a protein sample. This denatures tertiary structure and gives proteins a negative charge, allowing them to be separated by molecular weight using gel electrophoresis
Describe isoelectric processing
Proteins placed in gel with pH gradient
Will migrate to a pH which matches their isoelectric point
Describe 2D page
After isoelectric processing the gel is turned 90 degrees allowing proteins with the same pI to separate into different weights
What can be measure d using enzyme assays? Give an example of the clinical relevance of this.
Enzyme activity levels
An indication of whether an enzyme is present at normal levels
Test for enzymes that shouldn’t be there, e.g. Alanine transaminase shouldn’t be in the blood - indicator of liver damage
Under what conditions are enzyme assays performed?
Optimal pH, temperature and ionic strength
Including appropriate ions and cofactors
Describe western blotting, what technique usually precedes it?
Follows SDS page
Proteins transferred to nitrocellulose membrane and identified by conjugating with labelled antibodies
How are immunoassay carried out and what can they detect?
Antibody is immobilised on a slid support
Solution to be assayed is applied
Second antibody conjugated to an enzyme binds to the antibody-antigen complex
Binding of the second antibody is measured by assaying for the enzyme
Can detect protein concentration
Describe the process of southern blotting?
Fragments from electrophoresis are transferred using nylon. These fragments can then be hybridised with a gene probe to look for specific DNA fragments
What is southern blotting used for?
Mark unlabelled DNA from gel electrophoresis
Investigate gene structure e.g. Large deletions and duplications
Investigate gene expansions
Investigate variation, e.g. DNA fingerprints
What molecule is identified using northern blotting?
RNA
How can PCR be used for allele specific testing?
Use primers specific to the allele of interest to amplify specific allele
How can restriction enzymes be use for allele specific testing?
Use restriction enzymes with restricting sites near or within the allele - analyse the size of the fragments produced
How can DNA hybridisation be used for allele specific testing?
Use a DNA probe which is complementary to either the wild type or the mutated allele, see what binds.
What is a silent mutation?
A mutation which does not alter the amino acid sequence
What is a missense mutation?
Replaces one amino acid with another
What is a nonsense mutation?
Replaces one amino acid with a stop codon
What is a frame shift mutation?
Insertion or deletion which isn’t in multiples of three, so alters the whole amino acid sequence.
What is a spontaneous mutation?
A mutation that occurs randomly
What is an induced mutation?
A mutation that is cause by a mutagen, e.g. UV light
What is mismatch repair?
After replication any incorrectly inserted nucleotides are recognised and replaced with the correct base
What is excision repair? What are the two types?
Repair single stranded DNA damage caused by external damage. Base or nucleotide excision repair.
What happens if DNA is damaged to the point where apoptosis doesn’t occur or they grow uncontrollably?
Cells become cancerous
What is a oncogene?
Genes that control cell division in cancerous cells. When healthy they are known as protoncogenes
What is array comparative genome hybridisation used for?
Screen for submicroscopic chromosome deletions with unknown locus
How is array comparative genome hybridisation carried out?
An array of DNA probes covering the entire genome are applied to the surface of a solid matrix
Patient DNA is labelled red and control DNA is labelled green
Equal amounts of patient and control DNA is hybridised to the probe array
Green areas show deletions
What ethical issues are associated with gene technology? (3 examples)
Insurance companies could charge people differently for life insurance
People could ask for abortions because they don’t want a girl/ginger/I’ll e.t.c child
Getting tested for a dominant condition - you’re family might rather not know
What constitutes chromatin? 4 things
DNA
RNA
Non-histone proteins
Histones
What is the difference between euchromatin and heterochromatin?
Euchromatin is actively transcribed - Pale in colour
Heterochromatin is tightly packed, dense and darker in colour as it is not actively transcribed
How many chromosomes in humans?
23 pairs - 46 chromosomes
Give 4 examples of numerical chromosome abnormalities
Polyploidy - a complete extra set of chromosomes e.g. Triplody
Aneuploidy - a number of chromosomes that isn’t a multiple of the haploid number
Monosomy - a loss of one chromosome
Trisomy- a gain of one chromosome
What are structural chromosome abnormalities?
Physical changes to one or more chromosomes
What is the difference between balanced and imbalanced structural chromosome abnormalities?
Balanced- doesn’t result in missing or extra genetic information
Imbalanced- does result in missing it extra genetic information
What types of single chromosome mutation are there? Hint: 5
Deletion Duplication Inversion Ring chromosome due to lost telomere Isochromosome - creation of two non identical chromosomes
What type of mutations occur between 2 chromosomes? 3 things.
Inversion - rearrangement to a non homologous chromosome
Reciprocal translocation - an exchange of genetic information between 2 non homologous chromosomes
Robertsonian translocation- rearrangement of genetic material between two chromosomes where 2 long arms combine and 2 short arms are lost.
What is a karyotype?
A picture set of metaphase chromosomes organised systematically into pairs.
How would you describe a woman with 46 chromosomes who has a missing segment of the p arm on chromosome 5?
46, XX 5p-
How would you describe a man with triploidy who also has some extra genetic information on the q arm of 10th chromosome?
69 XY 10q+
What 3 prenatal screening results may mean the patient requires karyotype screening?
Down’s syndrome
Family history of chromosome abnormality
Abnormal fetal ultrasound
What other 6 problems may lead to karyotype referral?
Malformations at birth Mental retardation at birth Abnormal sexual development Infertility Recurrent fetal loss Leukaemia
Describe FISH
DNA probes hybridised to target DNA in sample
Fluorescent probe shows locus of target DNA
Can be used to identify whole parts of chromosomes
