Nucleic Acids Flashcards
Describe Franklins’ and Wilkins’s investigation of X-ray diffraction
The wavelength of X-rays makes them particularly sensitive to diffraction by the particle in biological molecules such as DNA. If X-rays are directed at a crystal the diffraction occurs in a regular way because in crystal the particles are arranged in a regular repeating pattern. DNA cannot be crystallised but Wilkins succeeded in producing arrays of DNA molecules in an orderly enough fashion for a diffraction pattern to be obtained. Franklin developed a high resolution detector that produced sharp images of diffraction patterns from DNA.
What did they deduce about the structure of DNA from the diffraction pattern?
- The cross in the centre indicate the DNA is helical
- The angle of the cross shape showed the pitch
- The distance between horizontal bars showed that the turns were 3.4nm apart.
Describe Hershey and Chase experiment
They provided evidence that DNA is the genetic material
1. They used the T2 virus/phage which consists of DNA inside a protein coat
2. When the virus E coli infects cells only the viral DNA enter the bacterium the protein coat does not
3. DNA contains Phosphorus only and protein contains sulphur
4. Two strains of T2 were prepared- one with the DNA radioactively labelled using 32P and the other having its proteins labelled with 35S
5. Each infected E. coli
6. Mixture was agitated in a high speed mixer and centrifuged to separate the solid pellet containing the bacteria from the liquid supernatant.
7. A Geiger counter was used to locate the radioactively
8. When 32P was used, most of the radioactivity was detected in the pellet. This is because T2 injects its DNA into the bacterium
9. When 35S was used, most of the radioactivity was detected in the supernatant. Agitation shakes most of the viral protein coats off the outside of bacteria.
Describe nucleosomes
The 8 proteins consist of 2 copies of 4 different types of histones
Another histone protein called H1 binds the DNA to the core
A short section of linker DNA connects one nucleosome to the next
The 8 histones in the core have N- terminal tails that extend outwards
The association of histones with DNA contributes to supercoiling. During condensation of chromosomes in the early stages of mitosis and meiosis the tails of histones in adjacent nucleosomes link up and pull the nucleosomes together
Chemical modification of the histones’ N- terminal tails is involved in regulating gene expression
What do nucleosomes do?
Facilitate the packing of large genomes of eukaryotes into a small space within the nucleus
What are tandem repeats?
Repetitive sequences found in centromeres and heterochromatin. They are regions of DNA that consist of repeats of the same sequence. This includes variable number tandem repeats (VNTRs) and short tandem repeats (STRs) which are often used in DNA profiling because the number of repeats a given locus varies between individuals so different individuals generate unique DNA profiles
Distinguish between gene and repetitive sequences
Gene- small proportion of genome, repetitive sequence- high proportion of genome
gene- low rate of mutation, repetitive sequence- high rate of mutation
gene- once in genome, repetitive sequence- occurs many times in genome
gene- translated, repetitive sequence- not translated
gene- similar between individuals (not using in DNA profiling), repetitive sequence- varies between individuals (used in DNA profiling)
gene- long unique sequences, repetitive sequence- short repeating sequence
Give an example of a gene and a repetitive sequence
gene- exons of genes
repetitive sequence- telomeres
What is gel electrophoresis?
Method of separating fragments of DNA according tot their size
Describe gel electrophoresis
The molecules to be separated should be charged. The samples are placed in wells in a gel. The gel acts as a molecular sieve. The gel is immersed in a conducting fluid and an electric field is applied. The charged molecules move towards the pole with the opposite charge so DNA molecules, move towards the positive pole.
Small fragments move faster than large ones so they move further in a given time. The thickness of the band indicates the quantity of the DNA. The thicker the band the more the DNA.
What is DNA profiling?
A technique used to identify individuals by their DNA
What does DNA profiling do?
The DNA profiles of 2 samples are compared. The technique is based in the fact that it is extremely unlikely that 2 individuals will have the same pattern of bands
Describe the method of DNA profiling
Sample of the DNA to be tested and reference sample of DNA are obtained
DNA loci that vary considerably between individuals are selected and copied by PCR
The amplified DNA is cut into fragments using restriction enzymes
The DNA fragments are separated by gel electrophoresis according to their length
This produces a pattern of bands that is unique to the individual
They are then compared
Alternatively to the DNA loci, STR loci or VNTR loci may be used as it is very unlikely for two individuals to have the same number of repeats at each of these loci and therefore the same band pattern.
What are uses of DNA profiling?
Forensics investigation
In paternity testing
To check whether two organisms are clones
In genealogical investigations
What is DNA/gene sequencing?
Technology that finds the base sequence of a piece of DNA/gene
What is the method of DNA sequencing (Sanger sequencing)?
Dideoxyribonucleotides contain dideoxyribose instead of deoxyribose so they do not have the -OH group on C3 which is necessary for forming the phosphodiester bond
When dideoxyribonucleotide are incorporated into the new DNA they stop replication because there is no site where another nucleotide can be added
Single- stranded copies of the DNA to be sequenced are placed into Eppendorf tubes will all materials needed for replication, deoxyribonucleotides and DNA polymerase
Very small quantities of dideoxyribonucleotides are also added
4 replication mixtures are set up each containing normal deoxyribonucleotides plus one of the four dideoxyribonucleotides (ddATP, ddTTP etc.)
The fragments are separated by length using gel electrophoresis
The base sequence can be determined by ordering fragments according to length
What was the Human Genome project?
A project with the aim to find the sequence of the entire human genome
What did the Human Genome Project show?
That humans have approximately 23,000 genes and that most of the genome is not transcribed- originally called ‘junk DNA’
What are potential positive outcomes of the Human Genome Project?
Better understanding of genetic conditions/diseases
Study of similarities/differences between human populations
Study of human origins/evolutionary relationships with other species
Identification of conserved sequences between humans and other species
Production of pharmaceuticals based on DNA sequences
What in the histones in involved in regulating gene expression?
Chemical modification of the histones’ N-terminals tails
Describe non-coding sequences
most are neither transcribed nor translated but some are transcribed but not translated.
What do non-coding sequences include?
- Sequences involved in the regulation of gene expression either by promoting or repressing the transcription of an adjacent gene eg. promoters, enhancers, silencers
- Introns
- Sequences that guide the production of tRNA and rRNA
- Telomeres
- Tandem repeats
Describe how introns are non-coding regions
Non-coding sequences within genes which are removed from the mRNA before it is translated. Introns are also involved in gene expression as they may contain negative or positive regulators of gene expression and involved in post-transcriptional mRNA processing and splicing
Describe how telomeres are non-coding regions
Telomeres are repetitive sequences at the ends of eukaryotic chromosomes. The enzymes that replicate DNA cannot continue replication all the way to the end of the chromosome. Sacrificing the repetitive sequences in telomeres serves a protective function.
Describe how tandem repeats are non-coding regions.
Tandem repeats are repetitive sequences of DNA found in centromeres and heterochromatin. The length of the repeated sequence can be anything from 2 bases to 60 or more. They include VNTR and STR which are often used in DNA profiling because the number of repeats at a given locus varied between individuals so different individuals generate unique DNA profiles.
What is gene expression?
Production of mRNA by transcription and then production of a polypeptide by translation.
What is a critical part of cell differentiation?
Regulation of gene expression.
Describe gene expression
Some genes are only expressed at certain times and/or certain cells and this is a crucial part of embryonic development as cell differentiation depends on differential gene expression.
In which levels does gene expression take place?
A the level of transcription, translation and well as post-translationally(in eukaryotes)
Describe the regulation of transcription by proteins that bind to specific DNA sequences
- Control of transcription/gene expression involves specific base sequences in the DNA and proteins that bind to these sequences
- The promoter is a sequence located close to the start of a gene where RNA polymerase binds to initiate transcription
- The promoter itself is not transcribed, so it is a non-coding DNA with a function
Describe the additional step in prokaryotes in the regulation of transcription by proteins that bind to specific DNA sequences
RNA polymerase binds directly to the promoter and starts transcribing. Repressor proteins can also bind to the promoter and prevent transcription
Describe the regulation of transcription by proteins that bind to specific DNA sequences in eukaryotes
- Proteins called transcription factors bind to the promoter. Some TR increase/promote transcription by facilitating binding of RNA polymerase to the promoter and some decrease/ inhibit transcription by preventing RNA polymerase binding
- Several TR are required. Some are activated by binding of a hormone or other chemical signal
- Regulatory proteins bind to regulatory DNA sequences and can increase or decrease the rate of transcription
- While enhancers and silencers can be distant from the promoter, other regulatory sequences are nearer to the promoter and binding of proteins to them also controls transcription. Regulatory sequences are also found within introns
- Eukaryotic genes are also regulated in response to variations in environmental conditions.
What are enhancers?
Regulatory sequences in the DNA which increase the rate of transcription when proteins (activator) bind to them
What are silencers?
Regulatory sequences in the DNA which decrease the rate of transcription when proteins (repressors) bind to them
Describe the regulation of transcription by modification of histones in nucleosomes
- Chemical modification of the histone N’-terminal tails is involved in regulating gene expression by controlling which sections of the chromosomes are condensed and which are decondensed during interphase
- Decondensed regions are transcribed because they allow access to transcription factors and enzymes that carry out transcription. Condensed regions are not transcribed because they are not accessible to transcription factors and enzymes
- These chemical modifications are reversible and include methylation, acetylation and phosphorylation
- Some modifications activate and some deactivate genes
- In decondensed regions, the H1 histone protein is removed so the binding of DNA to the nucleosome is loosened
Give an example of the regulation of transcription by modification of histones in nucleosomes
Residues of the amino acid lysine on histone tails bear a positive charge that binds to the negatively charged DNA to form a condensed structure. Histone acetylation neutralises these charges allowing a less condensed structure, thus promoting transcription
Describe regulation of transcription by DNA methylation
- Methylation of DNA inhibits transcription, so is a means of switching off gene expression
- Cytosine in DNA can be converted to methylcytosine by the addition of a methyl group (-CH3)
- The modification is enzyme-catalysed and only happens if there is guanine on the 3’ side of the cytosine in the base sequence
- Fluorescent markers can be used to detect patterns of methylation in chromosomes
- In some eukaryotes there is widespread methylation in parts of the genome
- The cells in a tissue have the same pattern of methylation and this pattern can be inherited in daughter cells produced by mitosis
- The pattern of DNA methylation changes during a lifetime and is affected by environmental factors
What has analysis of regulation of transcription by DNA methylation shown?
- patterns of methylation are established during embryo development and the percentage of CG methylation reaches a max at birth and then decreases during the rest of the life
- At birth, identical twins have very similar methylation patterns but differences accumulate during their lifetimes
Describe gene expression in terms of post- transcriptional modification.
Eukaryotic cells modify the mRNA after transcription, while it is still in the nucleus. Gene expression is also regulated at the post- transcriptional level eg alternative splicing thus in alternative splicing a single gene codes for more than one polypeptide.
Introns play additional roles in regulation of gene expression because they can carry positive or negative DNA regulatory sequences.
What is Epigenetics?
The study of heritable phenotypic changes that do not involve changes in the DNA sequence. Proposed to by Lamarck
What are explanations to epigenetics?
- Chemical modifications of chromatin that fix the pattern of gene expression. These are known as epigenetic tags/marks and these all contribute to epigenome
- Evidence that some epigenetic modifications are passed on to the next generation as specialised cells have their own epigenetic pattern.
