Download Genetics 3500 winter Test ii_ansers

1
Genetics 3500 winter 2016
Test ii
10 marks
The goal of the ENCODE project is to provide a detailed understanding of how
genetic information is organized on our chromosomes. This insight have been made
possible by the completion of the Human genome sequencing program in 2000 and
the development of newer high throughput DNA sequencing technologies. Describe
in detail what the ENCODE project has told us about how genes and how DNA
encoding elements are organized on the human genome.
See table in paper by Gerstein Expectation is that you enumeration several
examples and include a few words of explanation.
Should include:
Much of the genome is transcribed as NcRNA some of which overlap protein
sequences
Exons can be shared by unrelated proteins.
Introns can contain open reading frames of oother genes.
RNA editing so proteins do not reflect DNA sequence
Chromatin modification, Methylation of DNA and Histone modification affect gene
regulation (information not embedded in DNA sequence
Abundance of Transposable elements, some of which are still active
CNV affect gene expression and an important form of genetic variation.
Enhancer and silencers may be distant from genes that re regulated
Abundance of pseudogene some of which are transcribed Why?
Alternative splicing is very extensive. Proteins have an average of >5 splice forms.
Transplicing of transcript on different chromosomes makes some novel proteins.
Protein modification cannot be predicted from DNA sequence.
Rna genes suchas MiRNA and SNrna’s
2
6marks
Describe the application and significance of the Ames test.
The Ames test is used to determine whether metabolic products of a particular substance are
mutagenic
Salmonella strains that are his – and deficient in one or more DNA repair enzymes are used.
The compound is added to cells and liver extract and the mix is plated on his- media plates
Comp’d are mixed with liver enzymes because many compounds are not themselves mutagens but
become mutagens as a consequence of metabolism.
The bacteria cells that grow are revertants. The number of revertants is an indication of how
mutagenic the compound is and the number of mutants associated with a particular strain indicated
the type of DNA damage done by a particular mutagen.
4 marks
What are the potential consequences of chromosome breakage and rearrangement
events due to the activation of DNA repair systems
On rare occasions chromosome breakage will result in the inactivation of a specific
gene . That’s because the amount of chromosome space occupied by genes is pretty
small (1-2%).
More frequently chromosome breakage and rearrangements are likely to move
genes so that their expression is affected by other enhancer sequences. The
example the text referred to is this one.
https://en.wikipedia.org/wiki/Philadelphia_chromosome
3
6marks
Describe the principle of PCR and indicate why this technology warranted the
awarding of a Nobel Prize in 1994.
Define.
PCR is a technique that enable the amplification of specific Piece of DNA form a
template DNA that is determined by ss DNA primers that flank the region that you
wish to amplify.
Define components.
Primers, nucleotides, heat stable DNA polymerase, buffer and PCR machine aka
thermocycler.
Explain role of heating step, the n what happens when sample is cooled and primers
bind specifically.
Consequence of repeated cycles, …..logarithmic amplification of DNA.
4
4 marks
For decades “Lamarkism” aka the theory that Giraffes acquired long necks because
they need to reach foliage at the top of trees (inheritance of acquired characteristics
) was derided as junk science. We now now know that the effects of some
environmental exposures can be heritably passed on to the progeny for at least one
generation. This is called epigenetic inheritance. What is the molecular basis for
this kind of inheritance?
Methylation of DNA specifically the cytosine residues.
Modification of histone proteins methylation (decreases access of TF to DNA)
Deacetylation.
Chromatin remodeling…..shifting the position of histones along the DNA to provide access of TF to
promoter regions.
4 marks
What are the common feature of a plasmid vector used in recombinant DNA research?
See plasmid images on web.
Restriction sites
Promoter to control rate of transcription of the inserted gene product
Termination sequence
Selection marker
Antibiotic resistance.
5
Answer one of the following two questions for 6marks
(i)
What role does Rb play in the regulation of the cell cycle and how the
does p53 regulate the cell cycle when DNA damage is detected?
(ii)
What is the purpose of the RAS pathway? How does it work?
More details are expected in your essay. You may find it helpful to include an
image and describe all the point that the figure is intended to show the reader.
Rb is synthesized and binds to the transcription factor E2F. Phosphorylation
of RB by specific cyclinCDK complex (cyclin and cyclin-dependent kinase)
results in Rb releasing E2F. This protein is then able to act as a TF to promote
synthesis of key genes needed to replicate DNA.
When DNA damage is detected, P53 acts as a TF to promote the synthesis of
several genes, one being p21. P21 prevents the cyclin CDK’s from
phosphorylating Rb. For addiitonal background, Google images of p53 and
Rb , cell cycle
RAs mediates a signal from outside the cell that results in the promotion of
cell division. Binding of a growth factor to a growth factor receptor on the
membrane triggers activation of RAS. In its inactive state RAS has GDP
bound to it . RAS is activated when GTP is bound to RAS displacing the
GDP. Activation ras then activated other proteins such as RAF leading to a
signal cascades that leads to replication of DNA and cell division.
6
7
6 marks
Multidrug resistance in cancer therapy results from the increased expression of specific
membrane pumps that prevent the accumulation of a wide variety of chemotherapeutic
agents in the cancer cells. Explain how the technique of real time PCR could be used to
determine the levels of MDR-1 in a biopsy of tissue comparing gene expression in tumor
cells and normal tissues, in order to assess a patient’s prognosis.
***Start with a short definition of PCR
PCR is technique that results in the logarithmic amplification of a specific sequence of
DNA that is determined by short primers which flank the region of DNA to be amplified.
***First mRNA is extracted from tumour and normal cell tissues and converted to cDNA
using reverse transcriptase.
*** In real time PCR the amplification of ds DNA is measured after each cycle using a
fluorescent dye. (more DNa that is made results in more fluorescence)
**** The amount of fluorescence generated using primers for MDR-1 in a sample is
compared to the amount of fluorescence generated using primers for a gene that is
assumed not to change its expression under most conditions.
***The relative expression of MDR, relative to the expression of the control gene in each
sample is used to assess whether MDR-1 is up-regulated or down-regulated.
8
6 marks
Describe how RE enzymes ligase plasmid vectors and E coli Bacteria can be used to
clone DNA fragments isolated from a DNA sample from the Zika virus
Define the components used:
a. Restriction endonucleases. What do they do, what makes them specific
cutters?
b. Ligase What does it do? How is this enzyme used in conjunction with RE to
make a recombinant molecule.
See below:
Process:
1. RE is used to cut the plasmid and the DNA source (zika virus) to create sticky
ends.
2. Plasmid +DNA is mixed and ligase is added
3. Transformation of plasmid and selection of colonies of plasmids with inserts.
4. Only colonies with the transformed plasmid grow (why?)
9
6 marks
You have a choice of either using the technique of microarrays or RNAseq to study gene
expression in a ripening tomato fruit.
Chose one of these techniques ,briefly explain what information it can give you and
generally outline how it can be used to help understand this biological process.
RNAseq
*RNAseq is a technique in which the MRNA from a tissue is extracted and converted to
cDNA with reverse transcriptase.
Sequencing of cDNA enables estimation of relative expression of all mRNA reads.
Can see expression of ncRNAs, protein coding genes and their alternative splice forms
Upregulation of key genes can suggest a particular molecular mechanism that can be
experimentally tested.
Short version of the process (Not needed)
cDNA is sheared into smaller pieces, sequencing primers are ligated onto the fragments
sequencing primers enable the sequencing of fragments generating millions of reads
reads are assembled back into mRNAs and aligned to the genome.
Number of reads of each gene type is calculated.
Microarrays.
Microarray chip consists of a series of spots in which ss DNAs are attached that are
capable of binding to a portions of the sequence of each gene from the organism.
mRNA is isolated form tissue converted to cDNA and tagged with a fluorescent dye.
cDNa is hybridized to the chip ad the amount of fluorescence at each spot is a measure of
the number of copies of mRNA for that gene in the sample
Upregulation of key genes genes can suggest a particular molecular mechanism that can
be experimentally tested.