the human genome and disease Flashcards
1
Q
what is model one of higher order chromosome packaging. supercoiling nucleosome
A
10mn nucleosomes supercoil to form a 30nm superhelix (6 nucleosomes per turn) this forms loops averaging 300nmin length
2
Q
what are the features of a chromosome
A
· Centromere - a region that is highly repetitive, binds two sister chromosomes together. Either in the center or towards the top
· Sister chromatid
· Homologous chromosomes - a pair of chromosomes (similar to each other)
· Telomers - end of chromosomes, specialised to protect the chromosomes from being degraded. (may be able to prolong aging if you prevent telomeres from shortening)
3
Q
What is idiotype?
A
the specifici banding pattern of a single chromosome
4
Q
what is the long and short arm of the chromosome
A
P is short
Q is long
5
Q
features of prokaryote genomes
A
Single circular genome (haploid)
Sometimes carry episomes (aka plasmids: separate from the main genome)
Small and compact
6
Q
what are general features of stem cells
A
can divide without limit, allows for growth / development and replacement, · Divide into either another stem cell or differentiate into a new cell type called a terminally differentiated cell.
7
Q
which cells cen be replaced or repaired
A
- endothelial lining, New olfactory neurones, spermatozoa, gut lining, production of blood cells. permenant cells cannot be replaced
8
Q
what are topologically-associated domains
A
(sub-territory within chromosomes) is a self-interacting genomic region that organizes based on activity of chromatin interactions, splitting into active TAD compartments and Repressed TAD compartments
9
Q
how does the amount of methylation and acetylation determine where a chromosome is open or closed
A
More methyl groups compared to acetyl the region is open and active, fewer methyl groups and more acetyl groups mean it is closed so is no longer able to transcribe so repressing. balance
10
Q
what is the central dogma of molecular biology
A
DNA -> RNA -> Protein, from DNA to RNA is non coding, RNA to Protein is coding
11
Q
what are the gaps between genes within the DNA called
A
intergenic DNA
12
Q
what is the use of alternative sigma factors?
A
different sigma unit at different temperatures
13
Q
what are transcriptional regulators (transcription factors)
A
proteins that bind DNA, repressors - bind in operator site -35 -10 blocking RNA polymerase, Activators bind upstream from -35-10 stablising interaction between RNA pol and DNA
14
Q
if glucose levels are high what is the effect of the CAP protein in the lac operon?
A
- High glucose levels mean levels of cAMP stay low
2. CAP protein remains inactive so there is no activation of lac operon
15
Q
low lactose, low glucose
A
- operon off. cAMP levels are high and bind CAP to DNA. Lac repressor is still active so it binds to operator and RNA polymerase cant bind so no transcription occurs.
16
Q
what is the process of cell line packaging
A
a. The packaging cell line contain an integrated retrovirus / provirus same structure as retrovirus but without the packaging signal.
b. RNA polymerase binds to the LTR and there will be transcription of the viral DNA to RNA
c. Some RNA will be translated into proteins (viral proteins)
d. Capsid protein aggregate together to form outer capsid shell
e. Reverse transcriptase will be packed in this shell. And will look for viral RNA to package. As there is no packaging signal the capsid remain in the cell.
f. The retroviral vector with therapeutic DNA can be introduced into cells using liposomes. And migrate to the nucleus.
g. RNA polymerase will bind to the LTR and will transcribe DNA to RNA which will then contain the packaging signal
h. This is then packaged in the waiting capsid shells, and migration to the cell surface and budding off occurs.
17
Q
what was the ex vivo approach used to tranfer fucntional ADA gene back into patient
A
- Collected T cells form ADA patient and created retroviral particles with a functional ADA gene
- Retroviral particles were used to infect patients T cells in a cell culture
- Integration give some ADA+ cells and these cells are selected.
- Transfused back into ADA deficient patient. Expression of introduced ADA gene can overcome ADA deficiency. After 2 years near normal T cell count, with 25% of cells expressing ADA gene.
18
Q
what are general features of lentiviruses
A
Another type of retrovirus
· The most infamous lentivirus causes AIDs
· RNA genome (ssRNA -9kb)
· Can integrate into the host cell genome
· Can infect both dividing and non-dividing cells - big advantage
19
Q
what is approach one: Direct siRNA therapy
A
Synthesis of RNA oligonucleotides with sequence complementary for the target transcript
· Formation of a double stranded siRNA duplex
· Packaging into liposomes for delivery across the lipid bilayer and into cell.
20
Q
how is mature mRNA formed
A
RNA polymerase bind to the promotor region and both introns and exons are transcribed into RNA. The splicing occurs and the introns are removed from between the exons leaving a mature mRNA. Translation can then occur.
21
Q
how are SNPs caused?
A
by mistakes in DNA replication, Mutagen exposure - Usually no effect, very rarely phenotype
22
Q
what is an example of an X linked diorder?
A
Haemophilia - inability to form blood clots due to lack of factor VIII. Males are more effected as they gene is on the X chromosome, so females are carriers.
23
Q
what are the models of tumour evolution?
A
Linear evolution, Clonal separation (selection pressures), clonal competition (antagonistic evolution), clonal cooperation (symbiotic evolution), parallel evolution (early branching but are selective to same mutagtion. (also drug treatment)
24
Q
What is complete dominance?
A
recessive allele in a heterozygote not expressed. F1 phenotype is the same as the dominant parent. F2 generation all 3:1 phenotypic ratio.
25
what are the types of gentic tests
· Prenatal test - detects mutated gene in embryo/foetus e.g. couple carrying cystic fibrosis
· Prenatal screen - Embryo/foetus are tested across population for an increased risk of a condition e.g. Chromosomal abnormalities - Downs
· New-born screen - population wide testing for treatable genetic disorders e.g. inborn errors of metabolism / sickle cell. Symptoms can be delayed with treatment.
· Diagnostic test - Confirms diagnosis based on symptoms e.g. A child with failure to thrive and frequent lung infections is tested for CF.
· Predisposition test - Detects presence of mutated gene e.g. Family history of breast cancer gives option of preventative surgery and frequent screening.
· Predictive test - detects highly penetrative mutation with early onset e.g. healthy person tested for Huntington's because parent has condition.
26
what causes changes in DNA methylation?
Parental body composition, maternal nutrition/ exercise, Pollution, social adversity (attentive mother) diet,body composition, exercise
27
how is cancer formed?
by oncogenesis or tumourogenesis, caused by mutations usually multiple.
28
what are the three classes of mutated genes based on function?
proto-oncogenes, tumour suppressor gene, caretaker genes.
29
what is an example of a tumour suppressor gene?
P53 on chromosome 17, o Binds DNA stimulating p21 protein that interacts with a cell division stimulating protein (cdk2 - regulates cell division). Mutant p53 can no longer bind DNA, so P21 doesn't provide a 'stop signal' for cell division and cell divide uncontrollably.
30
what has the potential to be treated by gene therapy?
Inherited diseases, cancers, infectious disease, immune disorders
31
what is autosomal recessive inheritance?
neither parent has charateristic phenotype. · Heterozygotes have the normal phenotype, so trait seems to skip generations. e.g PKU, Albinism, Sickle cell anaemia, Tay-Sachs disease.
32
what are the environmental influences n sex determination
o Temperature - dependent sex determination - crocodiles, turtles, lizards (egg incubation)Temperature sensitive enzyme aromatase converts androgens (male hormones e.g. testosterone) to estrogens (female hormones e.g. estradiol). Location-dependent sex determination - slipper limpet:
33
What is the X-O system?
Protenor system. Missing Y chromosome
§ Males has 13 chromosomes - 6 autosomal pairs and 1 X chromosome
§ Gamete without any sex chromosome is called a nullo gamete
§ Female has 14 chromosomes
§ Turner syndrome - sterile, lacking chromosome 23
34
what are the 2 types of sex determination in plants?
monoecious, Dioecious
35
How is DNA packaged ?
Nucleosomes,
| Higher order packaging - Model 1: supercoiling nucleosome, Model 2: radical-loop scaffold model.
36
what are 4 types of stem cell
Unipoitent, Multipotent, Pluripotent, Totipotent
37
what percentage of exome sequencing identifies gentic mutation
25-50%
38
what evolutionary traits have transposons played a crucial role?
```
· Neurotransmission
· Development and evolution of placenta
· Skin development
· Memory evolution
· Vision
· Starch digestion
· Immune system development
· Stem cell development
· X-chromosome silencing
· Chromatin structure regulation
```
39
what is a nucleosome
form the fundamental unit of chromosomal packaging. Organised into beads on a string, the gaps between the nucleosome are called linked DNA and the nucleosome if comprised of 8 histone molecules + 146 base pairs of DNA. One turn of DNA wrapped around a nucleosome is called a 7-fold condensation of naked DNA.
40
what is model 2 of higher order chromosome packaging: radical-loop scaffold model
o non-histone proteins bind at regular intervals to form loops in a rosette pattern. Non scaffold proteins are spread at regular intervals and loop the supercoils together
41
when is the chromosome most condensed
metaphase
42
what are the macro features of a chromosome
G-banding, idiotype, karyotype
43
What is G-banding?
staining metaphase chromosomes with Giemsa stain to give specific banding pattern. banding occurs bc some regions are more dense
44
What is a karyotype?
the number and visual appearance of the chromosomes in the cell nuclei of an organism or species. genome wide snapshot of individuals chromsomes
45
what is a genome
some of all genetic information in an organism
46
feature of eukaryotes genomes
Multiple copies of chromosomes within the nucleus
Circular mitochondrial and plastid DNA. Plants have chloroplast DNA
Lager genomes
47
the human genome project
Sequencing started in 1990 and was published in 2001 with 88% accurately. Was first completed to 99% accuracy in 2004. The 1% accounts form repetitive sequences, hard to identify sequences, or from centromeres.
48
who many letter are in our genome
2.85997 billion
49
define division
replication of cells, leading to growth
50
define determination
Internal mechanisms determine the identity a stem cell will become
51
define differentiation
Cell changes morphology and other characteristics becoming a specialised cell type, often post-mitotic
52
what are stem cells required for
growth and development, Replacement and repair
53
types of growth that stems cells undergo with examples
continuous e.g. Fish and Crustaceans, determinate - Growth stops in adults, stomatic cells replace in adults. E.g. Birds and mammals, Re-growth e.g. salamanders can regenerate a limb.
54
Define unipotent with example - smell
Becomes one cell type e.g. Olfactory basal cells (smell) Olfactory neurone in nose survives one to two months, replacement comes from the basal cells.
55
define multipotent
· - becomes several or many types e.g. Gut epithelium which can become Absorptive cells, Goblet cells, Enteroendocrine cells, Paneth cells.
56
what is an example of pluripotent stem cells
Bone marrow that creates all blood cells. Haematopoiesis: Pluripotent stem cell - myeloid / lymphoid progenitor stem cell - progenitor cells - blood cell types.
57
define totipotent with an example - early embryo
Can become all cell types, early embryo (blastocyst) e.g. when treated with retinoic acid insulin and thyroid hormone is produced.
58
what is an example of a totipotent cell line
P19 EC, treated with retinoic acid and fibroblast are produced. petri dish before treatment = neurons and glia are produced
59
what are the three sources of stem cells for stem cell therapy
Culture cells from early embryo, Adult stem cells collection and culture, De-differentiation adult cells in culture
60
what are features of cultured cells from early embryos? stem cell therapy
can be differentiated and implanted - gives a possibility of rejection as DNA from a different individual. · Use of nuclear transfer from adult somatic cells to make genetically identical embryos to the patient.
61
what are the advantages and disadvantages for adult stem cells collection and culture?
Adult stem cells are the ideal approach ethically and immunologically
· Not numerous
· Many not exist for all tissue types
62
What is de-differentiation?
nucleoplasm, exits in chromosomes territories. distributed in a non-random radial distribution
63
what determines chromosomal territories?
Gene content - dense=centre gene poor = outside, cell and tissue type variability, co-regulated gene clusters within the same territory, activity levels.
64
what are the regions that interphase chromosomes contain
Open - euchromatin is relaxed and typically transcribed,
| Closed Heterochromatin is compact and not transcribed
65
what des topology refer to
the open or closed state of the chromatin within the TAD.
66
what do TADs do
help separate heterochromatic regions from euchromatic regions by moving them relative to what chromosomes are int there, repressed TADs will be separated from active TADs.
67
What determines Heterochromatic or euchromatic state?
Histone modifications by acetylation, methylation or ubiquitylation
68
what are two histone modifying enzymes and their roles?
Histone Acetyltransferases (HATs), transfer acetyl groups onto histone tails, Histone Methyltransferases (HMTs), transfer methyl groups onto histone tails
69
What are cis-acting elements?
Telomeres - the very ends of chromosomes
o Centromeres - specialised DNA linking sister chromatids
o Promoters/enhancers - sequences that activate expression of nearby genes
o Silencers - sequences that repress expression of nearby genes
o Insulators - sequences that prevent more distal elements from affecting the expression of nearby gene
70
what are trans acting elements?
Gene - smallest unit of hereditary coding for a gene product (RNA/protein)
71
what percentage of DNA is coding (codes for protein)
1.5%
72
what are coding elements in DNA
enzymes, structural proteins, regulatory proteins, signalling molecules
73
what is exome sequencing
sequencing of RNA molecules made by the cell that are specifically making protein
74
What is a gene?
the entire DNA required to direct protein sysnthesis
75
size of human genome and number of genes
3200Mb and 31,000 genes
76
what is gene density
number of DNA per mega base of DNA 10^6 (humans 9genes/mb)
77
what is the gene structure
5' control/promotor region - determines how activity the gene is, so how much RNA and how much protein is produced. This is the region where RNA polymerase binds, and this enzyme catalyses transcription and makes the RNA.
· Coding region - part of the gene that codes for the gene, start with start codon ATG and ends with either TAG, TAA, TGA which are the stop codons.
· 3' Control region - Involved in the termination of transcription.
78
how does transcription of a gene occur
1. RNA polymerase binds to promotor region
2. RNA polymerase initiates transcription and is labelled +1 all bases before this are -ve (negative bases) and all down stream (i.e. what is being transcribed) are +ve positive.
3. Transcription occurs
4. Transcription is terminated at the terminator, which is within the 3' control region
5. Ribosomes bind to the RNA at the start codon and being translation until the stop codon to produce the protein.
79
what is the transcriptional control in terms of protein expression?
· More protein required - mRNA synthesis increases - protein synthesis increases
· Less protein required - mRNA synthesis decreases - protein synthesis decreases
80
what are mechanism for transcriptional control in bacteria?
Constitutive expressed genes - switched on all time (houskeeping)
Regulated genes - can be on or off e.g respond to enviromental stress
81
what are the sequences of bacterial promotors
-35 (TTGACA/AAGTGT), 1. -10 (TATAAT/ATATTA) also known as pribnow box
82
where does RNA polymerase bind to initiate start of transcription
between -35 and -10
83
what are the two features of bacterial RNA polymerase?
Exists as core enzyme -the subunits 2 alpha, beta, and beta prime subunit, lossly binding to DNA. Holoenzyme - ) Contains 2 alpha subunits, Beta subunit, Beta prime subunit, Sigma subunit The sigma ensure the enzyme recognises the -35, -10 of the promotor sequences and binds strongly
84
what is the mechanism of tracription?
1. RNA polymerase unwinds the two strands of DNA (top strand is the coding strand and the bottom is the template strand)
2. It Synthesises a complementary RNA copy of the DNA template stands.
3. The product is an RNA transcript that I equivalent to the coding strand.
4. RNA polymerase moves down the gene in the 5' to 3' direction and rewinds the DNA it has transcribed.
5. When it reaches the terminator within the control region the enzyme dissociated from the DNA and the RNA is released.
85
what are two mechanism of control? in transcription
Use of alternative sigma factors, Transcriptional regulation (positive - transcriptional activators, or negative - transcriptional repressors)
86
what are the three downstream genes of Lac operon?
· Lac Z - encodes for Beta galactosidase, an enzyme which cleaves lactose into its component sugars
· Lac Y - encodes for permease, which transport lactose into cells
· Lac A - encodes for transacetylase, which covalently modifies lactose
87
What are operons?
regions of DNA that contain clusters of related genes, made up of promotor operator and mulitple related genes.
88
how is Lac operon regulated
Lac repressor - (negative) binds to operator site that overlaps promotor region blocking RNA poly binding, CAP activating protein (positive)The lac repressor is encoded for by the Lac I gene that lies upstream and has own promotor which is always on so lac repressor is always produced
89
what happens when lactose is present
1. Lac I gene produces the lac repressor, but a biproduct of lactose called allolactose is also present.
2. Allolactose binds to the lac repressor inducing a conformation change forming a repressor-allolactose complex that inactivates the repressor.
3. RNA polymerase can then bind leading to transcription of the genes and the proteins for lactose breakdown can be produced.
90
what happens when lactose is absent
1. When lactose is absent there is no requirement for the operon to be on.
2. As the lac I gene is always on the lac repressor is always produced and so it binds to the operator site.
3. This block the binding of RNA polymerase, so no transcription occurs.
91
if glucose levels are low what is the effect of the CAP protein in the lac operon?
1. Low glucose levels lead to an increase in cAMP (secondary messenger)
2. cAMP bind to CAP protein and induces a conformational change
3. CAP now bind upstream of RNA polymerase and interacts with it to give transcription.
92
high lactose, low glucose
operon needs to be turned on. Allolactose is present and so is high levels of cAMP so the CAP protein can bind to the DNA. (binding of RNA polymerase to promotor facilitated by CAP)
93
high lactose, high glucose
operon off. Allolactose is present so lac repressor is inactive. cAMP levels are low, so CAP protein stays inactive. Little or no transcription.
94
low lactose, high glucose
operon off. Lac repressor is active so binds and low levels of cAMP so CAP stays inactive.
95
what are the viral vectors for gene transfer
· Retroviruses
· Adenoviruses
· Lentiviruses
· Adeno associated virus
96
what are general features of viral genomes
· ssRNA ssDNA
· dsRNA dsDNA
· 7kb to 200 kb
97
what is the structure of an retrovirus (ssRNA)
```
· LTR - Long terminal repeat, contain a promotor, enhancer (start for gene expression) and polyadenylation sequence (at end).
· Ѱ - packaging signal
· GAG - capsid proteins
· POL - reverse transcriptase
· ENV - envelope proteins
LTR at end
```
98
How does the virus infect cells: life cycle of retrovirus.
1. ssRNA and reverse transcriptase is surrounded by capsid and envelope. Retroviruses use multiple receptors in the surface of cells to attached to them and the lipid bilayer fuses with plasma membrane so virus can enter into cell. Caspid is removed.
2. Reverse transcriptase then converts single stranded RNA into a double stranded DNA copy.
3. The DNA can randomly integrate into the genome of the host cell, so it gets passed onto daughter cells when cell division takes place.
4. RNA polymerase of the host cell bind to LTRs initiating transcription of the viral DNA and viral RNA is produced.
5. RNA is translated to produce, Capsid proteins, envelope proteins or reverse transcriptase.
6. When the capsid proteins are made they aggregate to form an outer protein shell, which then packages the reverse transcriptase, and starts looking for viral RNA to package.
· Viral RNA is identified as it contains a packaging signal
7. It will then migrate to surface of the cell and bud off and get covered in a lipid bilayer forming the envelope.
99
MoMLV (Moloney Murine Leukaemia Virus).
1. Isolating viral RNA
2. Reverse transcriptase is used to convert RNA into DNA
3. Then restriction enzymes can be used to manipulate the genome and start genetic engineering by cutting out GAG, POL, ENV and insert therapeutic DNA.
4. It can be packaged into retroviral particles using a packaging cell line.1. Retroviral vector particles contain therapeutic RNA, the fuse with the plasma membrane of patient's cells and the capsid will enter.
2. Once its enters the capsid is removed
3. Reverse transcriptase copies RNA into double stranded form
4. DNA integrates randomly into the genome of the patient's cells
5. The RNA polymerase will bind to the LTR and transcription and translation occurs to produce protein. Log term expression has been achieved.
100
what are most retrovirus vectors based on
MoMLV (Moloney Murine Leukaemia Virus).
101
what is an example of retroviral vectors being used
1990 - treat ADA deficiencyo Defective adenosine deaminase (ADA gene)
o ADA encodes for a gene in the purine salvage pathway, leads to accumulation of dATP which severely affects T lymphocytes. Severe combined Immunodeficiency (SCID),
102
what are the advantages of retroviral vectors
· High efficiency of gene transfer
| · High levels of expression
103
what are the disadvantages of retroviral vectors
· Max insert size 7-7.5kb
· Only infects dividing cells
Toxic? Insertional mutagenesis
104
why are lebtiviruses safer than retroviruses
· Engineered to remove the strong promotors and enhancers in the LTRs
· Replaced with weaker promotors = less chance of potentially drive high expression of an oncogene
· Are less prone to integration next to transcriptional start sites (but still little control over where they integrate)
105
What is adenovirus?
DNA virus
| · Genome is 36kb of ds DNA
106
what isthe life cycle of adenovirus
1. Early genes are expressed E1a and E1b genes
2. Expression of E1-E4 followed by viral DNA replication
3. Expression of late genes
4. Packaging of new virus particles and cell lysis
107
what are first generation adenoviral vectors?
: E1- and E3 removed to allow insertion of transgene and to stop viral replication. Low level of transcription from viral genes which led to innate host response. Cloning capacity 7.5Kb
108
what are second generation viral vectors
all early genes deleted because viral proteins encoded by these genes wwre shown to induce mist of the host immune reposne. These vectors have decreased toxicity and prolonged gene expresssion. Cloning capacity up to 35Kb
109
what are disadvantages of adenoviral vectors
Adenoviral vectors do not insert the gene into the host chromosome
· Limited to only temporary protein expression
· Doesn't work well on dividing cells
· Requires repeat treatments
110
what are features of gene silencing
Advantageous in a situation where the disease is caused by a gain of function mutation., · Strategy: selectively inhibiting the mutant gene from being expressed.
· Utilise a naturally occurring mechanism of gene silencing that is called RNA interference = RNAi
· RNAi is a mechanism of gene silencing used by a variety of different organism: animals, plants, fungi.
111
what is RNAi
a natural pathway of gene silencing and a defence mechanism that protects cells, its triggered by double stranded RNA
112
what is the process of gene silencing with RNAi
1. Ribonuclease: Dicer cuts dsRMNA into siRNA (short interfering RNAs - smaller fragments about 21 bp long)
2. siRNA are recognised by a protein called RISC (RNA-induced) silencing complex which degrades on of the RNA stands in the duplex
3. The remaining siRNA strand and RISC complex then seeks out RNA transcripts that have homologous and complementary sequences leading to the destruction of any transcripts that contain the same nucleotide sequence as the siRNA.
4. Cleaved products are then broken down by exonucleases within the cell
113
what is approach 2: short hair in RNA-mediated siRNA therapy
```
· shRNA = short hairpin RNA
· Gene with inverted repeats
· Transcribed in the nucleus
· Formation of a hairpin = double stranded RNA
Dicer cleaves to create siRNA
```
114
what are the disadvantages of RNAi
· Often the target gens is 'Knocked down' instead off 'Knocked out' because complete gene silencing is difficult to achieve with this approach
· Risks of off-target effects
· Delivering sufficient RNA to target cells can be difficult
115
what are regulatory regions?
regions that do not code for a protein but govern whether the gene next to it is going to go up or down
116
what is F-syndrome
a region within a non-coding portion of DNA that disrupts a TAD, causing a disruption in the three-dimensional structure of chromatin within the DNA leading to birth defects.
117
what are non-coding elements of the genome?
```
Cis acting (not transcribed) o Enhancers
o Silencers
o Telomeres
o Centromeres
o Insulators, Trans-acting (transcribed) o Regulatory RNA (short and long)
o Structural RNAs (rRNA and tRNA)
```
118
what are the two non coding structural RNA genes
tRNAs rRNAs
119
what are tRNA
main role protein translation. cytoplasmic or mitrochondrial
120
what are rRNAs
ribosomal RNA used during biogensis and translation.
121
what are the different classes of regulatory RNAs
Long Non-coding RNAs, Small Non-coding RNAs
122
what are the remaining regions of the genome?
Transposons, LINES, SINES. mutagenic and important for genome evolution, repetitive regions, intergenic regions
123
what is the difference between heritable mutations and transient mutations
H - occur in germ-line cells, T - occur in somatic cells
124
types of mutations?
Point mutations (insertion, deletions, substitutions), larger mutations (Insertions, duplications, Translocations, Inversions
125
what is the mechanism for point mutations
. A wild-type genome will undergo either DNA damage (natural process or mutation) or a DNA base sequence error (Natural DNA replication or occurs in presence of mutagen)
2. The DNA will try and repair it, however if the DNA is replicated before it is repaired a mutation occurs and becomes a permanent change.
126
what are chemical physical and biological mutagens?
P:· radiation and heat cause large double stranded breaks, C: Base analogue, Alkylating agents, intercalating agents, De-aminating agents, metal ions cause smaller mutations., B: virus, Bacteria or transposon cause large insertions
127
What are transposons?
mobile DNA elements that move from one place to another in the genome using enzymes called transpoase. region it jumps to becomes more euchromatic (open)
128
what are the two classes of transposons
· Class I (retrotransposons) - copy and pastes mechanism
| · Class II (DNA transposons) - Cut and paste mechanism leaving empty site
129
what is the human mutation rate
1 in a billion base pairs
130
what is the eukaryotic gene structure
· 5' Control region promotor
· Coding region - split into 3 exons with introns, non-coding sequences in-between
3' control region
131
what is splicing
a process by which a gene can form multiple proteins
132
what is the structure of the eukaryotic promotor ?
Highly conserved sequence of TATA at -30 upstream +1. RNA polymerase binds here. (TATA Box)
133
what are the three methods of transcriptional control?
transcriptional regulators,
| Chromatin structure, DNA methylation
134
what do transcriptional regulators do
Transcriptional repressors - Block RNA polymerase from binding so gene remains off.
· Transcriptional activators - RNA polymerase directs only a low level of transcription, so activators need to bind upstream of TATA box. Bind to USE (Up stream sequences) - found near TATA box or further away where it bends DNA to form a loop.
135
how does chromatin structure control transcription?
How tightly packed the histones are is controlled by transcriptional regulators through modifying histones., Transcriptional activators recruit histone acetyl transferases (HATs) which acetylates the lysine's in the tail to neutralise their charge. This reduces the affinity of the tail for DNA and opens up DNA allowing RNA polymerase to bind. Transcriptional repressors also modify histones by recruiting histone de-acetylase (HDACs) which removes acetyl group of histone unit which restore the positive charge and increases attraction to DNA preventing RNA polymerase from binding, shutting off transcription
136
what is DNA methylation
addition of a methyl group to DNA usually at C base, · DNA methyl transferases (DNMTs) - methylates DNA, recruited to genes by transcriptional repressors along with HDACs when long term repression (silencing) is required. low levels mean RNA polymerase can still bind
137
what i short term gene repression?
transitional repressors can Compete to block binding of RNA polymerase or transcriptional activators and recruit HDACs.
138
what is long term gene repression
recruitment of HDAC + DNMTS
139
how do different eye colours occur?
In normal circumstances the amount of gene product from OCA2 is determined by a nearby gene called HERC2 this will give dark/brown eyes. A single point mutation (SNPs) within HERC2 prevents HERC2 from making the right quantity of OCA2, this leads to blue eyes.
140
what are the types of genetic variants ?
SNPs - Single Nucleotide Polymorphisms (one nucleotide is changed)
· DIPs/InDels - Deletion-Insertion Polymorphism (1-100 nucleotides are inserted or deleted)
· SSR - Simple Sequence Repeats (1-10 bp units of nucleotides repeated)
· CNV - Copy Number Variants (10bp - 1mb same gene that just exists in different copy numbers
141
how are DIPs caused
· by mistakes in DNA replication, Repair - if within a protein coding region loss of a protein
142
how are SSRs caused
Very rare spontaneous event, multiply rapidly - If within a protein can lead to disease
143
how are CNV caused?
Unequal crossing over during Meiosis I - phenotypes can vary a lot
o An example of CNVs in the population, the number of copies of olfactory receptor genes (and types) determines ability to smell different smells.
144
what is the PAN-genome?
captures the whole genetic content of a species
145
what is the resilience project?
Some genetic diseases have distinct causes and effects. Some individuals might carry the mutation but do not show a phenotype (resilient individual), sequencing theses individuals genomes can be used to identify candidate 'resilience factors'. This could then help drug design or understand the disease etc. 589306 genomes analysed 13 resilient adults found.
146
what is natural selection?
A natural mutation arises that confers a selection advantage. It is then spread through reproduction in a population
147
what are examples of positive selection in humans?
Lactose intolerance - 80% of European descent have a mutation in the lactose gene, Malaria resistance - Beta hemoglobin gene (HBB) mutation in Africa due to high rates of malaria - confers resistance· Duffy antigen gene (FY) is present at a frequency of 100% in sub-Saharan Africa and 0% anywhere else.
148
what are the negative consequences of natural selection
Malaria resistance but sickle cell anaemia and thalassemia are high.
· Better able to control the immune response but high risk of inflammatory response.
· EGLN1 mutation gives better response to hypoxia but it also means blood cells aren't as healthy so you can get erythrocytosis and pulmonary hypertension.
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what are the two main causes of genetic disorders?
1. Chromosomal abnormalities
| 2. Single gene defects - monogenic diseases
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1. Chromosomal abnormalities
| 2. Single gene defects - monogenic diseases
Loss of a single chromosome - monosomy- not tollerated in mammals e.g. Cri-du-chat, loss of a small part of chromosome 5, severe mental retardation, physical abnormalities (malformation of larynx - cry sounds like cat) 1/50,000.
Gain in chromosomes - trisomy - e.g. down syndrome / trisomy 21 (maternal age linked as probability of rearrangement increases)
151
what are single gene disorders?
small changes in the nucleotide sequence. Point mutation, Insertion or deletion due to replication errors, Tautomerisation, deamination.
152
what is tautomerisation?
spontaneous structural alterations, they are capable f existing in two forms. e.g NH2 and NH or Ketone and Enol (C-OH)
153
what induces single gene disorders?
radiation, U/V (chemically alter bases), Intercalating agents (chemicals that insert between bases causing opposite strand to insert as base), base analogues - chemicals that look like bases and get incorporated
154
single gene disorders are classified by ?
if its on the X chromosomes or on one of the 22 autosomes. whether its recessive or dominant
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example of an autosomal recessive disease
Cystic fibrosis - defective CFTR gene which encodes a Cl- ion transported, chloride ions are not transported out of cell, causing mucus to build up outside - severe impairment of lung function. Mutation is Delta F508 which is the deletion of CTT which codes for Phenylalanine chromosome 7
156
example of an autosomal dominant disease
Progressive neurological degenerative disease caused by inheritance of defective Huntington gene on chromosome 4. Caused by trinucleotide repeat expansion, CAG sequence is expanded from 10-26 times to 37-80 repeats this gives a larger loop in the protein making it inactive.
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example autosomal dominant - inherited cancer/
example autosomal dominant - inherited cancer/
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what are ways of testing for gentic disorders
· Obtaining DNA sample from foetus by doing amniocentesis test in the 12+ week.
· Chorionic villus sampling (8th - 12th week)
· Preimplantation genetic diagnosis - during In vitro fertilisation (IVF) a single cell can be removed from the embryo and test it and put back disease free embryo.
· In adults blood samples can be taken or skin biopsy
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how are chromosomal abnormalities (1) and single gene defects (2)detected?
1. - detected as chromosomes can be visualised through a light microscope with Giemsa stain
2. using PCR to amplify DNA sequence
160
what are the two types of mutagen?
direct acting
| indirect acting - need modifying by cellular elements.
161
what are virally induced cancers?.
retroviruses that incorperate into the genome e.g. HTLV-1
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what are transmissible cancers?
tumours that can expand beyond a single individual. Cancer became maliganat in first animal known as founder, tumour cells disseminate into other animals and spread through the population.
163
what are examples of transmissible cancers?
. In dogs Canine transmissible venereal tumour (CTVT), Bivalve transmissible neoplasms (BTNs), Devil facial tumour 1 and 2 (DFT1 , DFT2).
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what are the features of Devil facial tumour 1 ?
· Tumour cells pass between individuals during biting behaviour
· Tumours are usually found on face and neck
· Highly aggressive - large population crashes (no evidence it transmits between species)
· Variable growth period of 3-12 months
· Derived from male devil
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what are Jean-Baptiste lamarcks 2 laws to explain evolution?
use and disuses (use=gain power ect), inheritance of acquired characteristics
166
define gene
a unit of inheritance often responsible for one trait
167
define genotype
the allelic hereditary constitution
168
what are the advantages of using peas in genetic crossing?
Several discrete traits that can be studied
· Self-fertilizing
· Numerous viable and fertile progeny
· Short generation time
· Can carry out crosses between different individuals by emasculation of another before pollen ripe and transfer of pollen from alternate parent
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what are medels four postulates and laws of inheritance
1. Principle of paired factors
2. Principle of dominance
3. Law of segregation or law of purity of gametes (homozygous to start)
4. Law of independent assortment
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what is medels first postulate
Unit factor in pairs For each character or gene locus, an organism usually inherits two alleles, one from each parent. Alternative versions of alleles account for variations in inherited characters (traits) these different versions are usually caused by mutation in the DNA code. E.g. character or gene locus for stem length TT (tall phenotype), Tt (tall), tt (short).
171
what is pure breeding?
homozygous tall stemmed plants crossed with a variety that produced short stems. All resulting seeds collected, produced tall plants.
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what is medels second postulate
if two alleles at a gene locus differ (heterozygous), the dominant one determines the appearance while the recessive on has no noticeable effect. Depending om traits, the uniform feature is either one of the parents' traits or its intermediate.
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what is medels third postulate / first law?
Segregation. At gamete formation the two alleles segregate randomly, one to each pole. If homozygous then alleles identical then so are all the gametes, but if heterozygous then 50% gametes will contain one allele type and 50% the other.
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What is dihybrid inheritance?
two genes on differnt chromosomes each with two alleles
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what is medels 4 postulate / second law?
Independent assortment. At gamete formation - meiosis, alleles on two or more different gene loci on different chromosomes assort independently form each other. In dihybrid inheritance each pair of alleles at a gene locus segregate independently of other pairs of alleles during gamete formation. Strictly speaking this is only for gene loci carried of different chromosomes (unlinked).
176
what is required for PCR?
Template DNA, DNA polymerase, Oligonucleotide primers, dNTPs (bases), Buffer
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what type of DNA polymerase is used in PCR
Thermus aquaticus (Taq) which has an optimum at 72 degrees and is relatively stable at 94 degrees. thermostable so doesn't need replenishing
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what is the process of PCR?
1. PCR occurs by heating DNA to 95 degrees to break hydrogen bonds to give two single strands.
2. Annealing of primers to DNA at 55 degrees Celsius. A pair of oligonucleotide primers (18 bases in length) sit at each end of the region that needs amplifying. One primer binds to bottom strand and one to the top.
3. At 72 degrees Celsius DNA polymerase extends the primer and copies the DNA strands. One cycle of PCR has now occurred.
4. After multiple cycles it is Primarily amplifying the DNA that lies between the oligonucleotide. Amplicons are the product of PCR after 30-40 cycles.
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how is PCR used to detect insertion or deletion mutations
· Primers are designed to bind to either side of the mutation
· Gel electrophoresis is used to separate the smaller PCR product using electrical current.
· Compared against control to detect insertion or deletion, in polyacrylamide gels 1 b.p. change can be detected.
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what is an example of an insertion or deletion mutation. and how do they show up on a PCR
I: huntington's - Expanded CAG sequence will get a normal and a slower band on PCR.
D: 3 bases pairs missing in CFTR gene, strong band futher down (smaller)
181
What is epigenetics?
a second code that influences genes and phenotypes, can be altered by enviroment. Process that induced long term stable changes in gene activity without a change in gene sequence.
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what are the major epigentic processes ?
histone modification,
| Non-coding RNAs, DNA methylation (linked to obesity depression ect.),
183
how do cancer cells differ from other cells?
less differentiated, more mitotic structures, less adherent, changes in metabolism, aneuploidy is common (chromosome instability)
184
What is metastasis?
spread of tumour cells to other parts of the body. through blood stream.
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how do mutation in the DNA lead to cancer ?
```
· Sustaining proliferative signalling
· Evading growth suppressors
· Activating invasion and metastasis
· Enabling replicative immortality
· Inducing angiogenesis
· Resisting cell death
```
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features of a benign tumour?
```
Expansive growth
Slow growing (in general)
Frequent stabilization of mutation
Mitoses - Rare + typical
Evolution Local
Non-Metastasizing
Favourable for spontaneous evolution
No recurrences of evolution after removal
```
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features of a malignant tumour
```
Infiltrating growth
Rapid growing (in general
Exceptional stabilization of mutation
Mitoses - Numerous + atypical
Evolution local + general
Possible metastasizing
Spontaneous evolution is always fatal
Common recurrences of evolution after removal
```
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how can a single gene transform cells to cause cancer in a culture
1. DNA from human tumour cells is added to mouse 3T3 cells
2. Cultured for 2 weeks
3. Focus of transformed NIH/3T3 cells growing among untransformed cells
4. Extract of DNA transformed new mouse cells and undergo a second's cycle of growth
5. Extract genomic DNA and found the 3T3 gene was responsible for the transformation.
189
what causes mutations that lead to cancer?
carcinogenic substances, viruses, inheritance e.g. BRCA1 & 2
190
what is the effect of mutation on proto-oncogenes compared to normal function?
normally promote cell survival and proliferation, mutation is gain of functions and allows unregulated cell survival and proliferation causing point mutation, chromosomal translocation and amplification. (dominant)
191
what is the effect of mutation on tumour suppressor genes compared to normal function?
inhibit cell survival or proliferation, mutation cuases loss of function allowing unregulated cell survival and proliferation. deletion, point mutation, methylation (recessive)
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what is the effect of mutation on Care-taker genes compared to normal function?
normally repairs or prevents DNA damage leads to loss of function allowing accumulation of mutations, deletion, point mutation, methylation (recessive)
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what is an example of a proto-oncogene
C-Myc, a transcription factor responsible for cell cycle regulation. C-Myc expression is highly regulated as its mRNA is short-lived and protein levels are low. Translocation of the c-myc gene occurs in Burkitt's lymphoma, resulting in enhanced expression. Located on chromosome 8, is moved near to antibody gen enhancer Chromosome 14. / · - Growth factors - HER2 in breast cancer. HER1 is a transmembrane growth factor receptor which upon ligand binding undergoes dimerization and transphosphorylation of their intracellular domains. This dimerization leads to a signal pathway that causes cell differentiation, Migration and cycle control, apoptosis, Angiogenesis. A V664E mutation promotes this receptor dimerization and activity.
194
what are the five classes of proteins are recognised as being tumour suppressor genes?
Intracellular proteins, Receptors for secreted hormones, Checkpoint control proteins, proteins that promote apoptosis, enzymes that do DNA repair
195
what is the effect of of the p53 gene in elephants?
they have extra copies of p53 so are more sensitive to DNA damage and apoptisis occurs more.
196
What do caretaker genes do?
repair damaged DNA, causing high mutation rates,· mlh1 and msh2, Involved in mismatch repair of DNA bases during DNA replication. Mutations in these genes increase the rate of point mutations in genes
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what are the 3 main types of mutation?
Aneuploidy - extra chromosomes, · more mutations, more chromosomal break points and more chromosomal fusions. Translocations - double stranded breaks that result in fused elememts from different chromosomesMay trigger abnormal cell growth by placing a proto-oncogene under inappropriate control of a constitutive promotor. Duplications - localised reduplication of DNA to produces as many as 100 copies of a given region (usually spanning hundreds of kilobases)
198
What is loss of heterozygosity?
Inherited mutation in tumour supressor gene gives predisposition for cancer when the remaining functional allele is lost this is LOH
199
what are the two mechanisms for loss of heterozygosity?
missegregation
| Mitotic recombination
200
what is the mechanism for missegregation?
1. A normal ad mutant allele exists with cell
2. At cell division an unequal segregation of the chromosomes occurs so one is haploid, and one has 3 chromosomes. Aberrant 3:1 segregation
3. After a further round of cell division, a heterozygous cell is formed and a homozygous for the mutant allele.
201
what is the mechanism for mitotic recombination
1. Heterozyogous cell for mutant allele (on tip of chromosome) undergoes chromosomal duplication
2. If recombination between homologous chromatids occurs the mutation crosses over
3. After a division this level as cell homozygous for a normal allele and one homozygous for a mutant allele.
202
what is hereditary retinoblastoma ?
Retinoblastoma is caused by mutation in the RB gene, Rb regulates cell cycle entry.
· Mutation in the RB gene can be hereditary but is recessive so only loss of the other allele makes the individual homozygous and tumours form in the retina.
203
what is the multi-hit model of tumourogenesis?
cancer isnt caused by a single mutation, consecutive mutations lead to an increase in proliferation and a class II adenoma (benign)
204
what is gene therapy?
describes any procedure intended to tret or alleviate disease by genetically modifying the cells of the patient. The method of gene therapy can include the use of DNA, RNA or Oligonucleotides.
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what are the two broad categories of gene therapy
Germ-line cells - genetically modified to produce a permanent modification that can be transmitted to descendants.
Somatic cells - modify specific cell that is not passed on and is only confined to the patient
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what are the 2 strategies involving target cells with the pathogenesis of diesease?
1. Modify the diesease cells to alleviate the disease through, gene augmentation, gene silencing, repair of mutant genes.
2. selectively kill the disease cells, directly or indirectly
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what is gene augmentation?
functional copies of the gene are added back into the cells that lack it, works well if the disease is caused by loss of function mutation. If the cell is damaged too extensively it won't work
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what is gene silencing?
the expression of the gene is selectively inhibited. Useful if the gene being expressed is harmful e.g. gain of function mutation. Can be achieved by turning off transcription of the harmful gene. Used to treat infectious diseases or oncogenes in cancer.
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what is repair of the mutant gene?
repair of the gene mutation to restore normal gene function or to minimise the effect of the mutation.
210
what is the direct way to selectively kill disease ?
· Targeting the cells with a suicide gene to code for a protein that is toxic to the cells. Or target a prodrug metabolising gene to the disease cells (prodrug has to undergo a change caused by a metabolising enzyme to convert from inactive to active) which kills the cells by toxic prodrug metabolites.
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what is the indirect way to selectively kill disease
mmune system is modified to enhance its response to the disease cell and initiate cell death. Disease cell are targeted with a foreign antigen gene that is recognised by the immune system or a cytokine gene can be transferred into the cells and will be expressed by diseased cells so that they get killed while non diseased cell (especially immune system cells) kill the diseased cells as directed by the cytokine.
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define gene transfer
the method of getting DNA into a patients cells in order to use this technique to treat disease
213
what are the 2 general approaches to gene transfer?
In-vivo (injected into tissue), Ex-vivo (cells are removed and modified before being returned)
214
what is a challenge to gene therapy in terms of the life span of cells being targeted?
short lived cells - divide to replenish, loss of added genetic material and therapeutic effectiveness.
Long lived cells - non-dividing but need gene to be efficiently transfered.
215
what are rules for gene transfer be?
Nontoxic
· Target the DNA to specific cells
· Introduce DNA into a cell in a transcriptionally active form
· Express the gene at a reasonable level for sufficient time
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what are the two main delivery vehicles for DNA?
1. Non-viral - liposomes, lipid1. encapsulate DNA and mix with the target cell and the liposome will fuse with the membrane allowing DNA to enter cell.
2. Viral- retrovirus, lentivirus, adenovirus adeon-associated viruses
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what are 2 types of non viral methods of delivery vehicles?
```
Cationic liposomes (most commonly used) liposomes contain therapeutic gene. They are taken up into the cell via exocytosis or Clathrin coated pits. DNA avoids degradation by lysosomes and is taken up into the nucleus. Once in the nucleus the gene need to be expressed.
· Compact DNA nanoparticles - relies on DNAs negatively charged phosphate groups and can bind to polycations which causes the DNA to become highly compacted. DNA is complexed with PEG-CK30 which contains 30 lysine residues and a N terminal cystine which is bound to PEG covalently. The DNA bind to PEG-CK30 and becomes compacted and condensed making it efficient in entering the cell and can carry plasmids with a capacity of at least 20kB
```
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genes need placing in a plasmid expression vector, to achieve high levels of expression. what is the structure of the expression vector?
· CMV (cytomegalovirus) promotor that's very strong and high levels of gene expression and works well in all eukaryotic cells.
· Multiple cloning site: contains sites for restriction enzymes e.g. EcoR1 & Sal1 where the gene is cloned in
· Intron with a SD (splice donor site) and SA (splice acceptor site), which say where the intron begins and ends. Intron is required as the genes need to be spliced. Clone in cDNA copy of the gene as it will have no introns.
· Polyadenylation site for efficient termination of transcription. Apalcillin resistance site and a bacterial origin of replication
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what happens when DNA enters the nucleus during gene therapy?
1. RNA polymerase of the cell binds to the CMVC promotor initiating transcription
2. The RNA copy gets cleaved and polyadenylated
3. Spliced and intron is removed
4. Transported to cytoplasm and ribosome will bind and scan through for the first AUG start codon for transcription.
220
what is the problem and solution to DNA entering the nucleus?
the plasmid tends to be very inefficiently transported into the nucleus because of its size, cant enter through pores in the nuclear membrane. So several approaches are used to get it in:
· Conjugating specific DNA or protein sequence to that are known to facilitate nuclear transport
· Compacting the DNA so that its smaller in size and can move through nuclear pores
221
what are uses of gene therapy for cystic fibrosis
Introduce functional CFTR gene back into the epithelial lung cells so it can be transcribed and translated to produce normal cells. 1989 used aerosols but DNA was ineffectively transferred by lipososmes.
222
what are the advantages and disadvantages of liposomes?
Non-toxic. But...
· Inefficient transfer of DNA to target cells
· Cannot target particular cell types
· Poor Expression
223
What is polygenic inheritance?
wo or more genes with two alleles having additive effect on phenotype. continous variation.
224
what are multiple alleles
a signle gene with 2+ alleles, different mutations in same gene result in range of allele genotypes. Only one allele is expressed at any time. e.g. drosophila eye colour
225
what is penetrance?
proportion of individuals showing expected phenotype - reduced penetrance can be due to epistasis, suppressors. 100% penetrance means all individuals express the phenotype e.g. Tays-Sach
226
what is expressivity ?
degree of expression of phenotype in an individual due to influence of other genes environmental effects e.g. temperature sensitive (Siamese cat), nutrition (lactose intolerance)
227
what are the environmental effects on phenotype?
Temperature, e.g. siamease cats Cs Cs coding for tyrosinase.
| Nutrition e.g. lactose intolerance, human Phenyketonuria (PKU)
228
what is human Phenylketonuria?
autosomal recessive. Defect in enzyme that metabolises phenylalanine, build up causing brain damage in children. Put children on low phenylalanine diet prevents mental retardation. Expressivity altered in individuals.
229
what is pleiotropy
when one gene influences two or more seemingly unrelated phenotypic traits
230
what are the three primary patterns of inheritance?
Autosomal recessive, Autosomal dominant, Sex-linked (X-chromosomal)
231
what is autosomal dominant inheritance?
Heterozygotes express mutant phenotype and 50% chance of passing on to progeny e.g. Huntington's, polydactyly.
232
what is sex linked inheritance?
trait is preferentially seen in males who are hemizygous. Females are usually heterozygous carriers but homozygous will show the trait. · Males get mutant allele from mother, Affected males transmit the mutant allele to all of their daughter but cannot pass it on to sons. (recessive mostly) e.g colour blindness
233
Asexual reproduction / parthenogenic reproduction
reproduction from a ovum without fertilization, mother provides a full set (2 sets of genes) e.g. Cloning aphids, plant vegetative propagation (spider plant). 80 species of unisex reptiles, amphibians and fishes for which males are no longer part of the reproductive process.
234
what is a hermaphrodite / bisexual / monoecious?
· individuals with both male and female reproductive organs. Gametes produced by single individual, may be self-fertile or require partner. Common in plants & Annelids, Molluscs, invertebrates
235
define Ghonochoric / unisexual / dioecious
male and female reproductive organs in separate individuals (humans)
236
how is sex determination genetically controlled?
Haplo-Diploidy, X-O system, X-Y sytem, Z-W system
237
what is Haplo-Diploidy
(common in bees and ants)
§ Non sex chromosomes
§ Males develop from unfertilized eggs and are haploid (drone)
§ Females develop from fertilized eggs and are diploid (queen and workers)
§ Males produces spermatozoa via mitosis
§ Queen produces unfertilized eggs via meiosis
238
What is the X-Y system?
Humans, 2 X and 1 Y leads to Klinefelter syndrome. Y Promotes male development in embryo, SRY gene on Y switch from female to male
§ Human / mice maleness - the SRY gene. In mice and humans, the presence if a proportion of Y chromosome is essential for initiating testis development
239
What is the Z-W system?
§ Birds and lepidoptera (butterflies) use a chromosomal system, however unlike the mammalian system males are Homogametic ZZ and females are heterogametic ZW
240
define monoecious
hermaphrodite flowers - so both male and female reproductive organs are produced on the same plant e.g. Lily, Maize.
241
define Dioecious
·Male and female flowers on separate plants e.g. XX-XY Meladrium (Campion), Date palm, spinach, Marijuana.
