Topic 7: Nucleic acids Flashcards
DNA as the genetic material
Hershey and Chase wanted to see what the genetic material was made of.
They used a virus that infects E. Coli to investigate whether genes were made of DNA or protein.
Prepared two strains of T2 virus, one had its DNA radioactively labelled with Phosphorus, and the other with Sulphur.
Mixed with E. Coli and centrifuged to separate the solid pellet with the bacteria and a liquid supernatant.
(T2 binds to the surface of E.Coli and injects its DNA into the bacterium)
High proportion of radioactivity with the bacteria in the pellet when Phosphorus was used.
High proportion of radioactivity in the supernatant when Sulphur was used.
The small proportion of radioactivity in the pellet can be explained by the protein coats that remain attached to the bacteria.
Strong evidence - genes being composed of DNA rather than protein.
Helical structure of DNA
If a beam of X-rays is directed at a material, most of it passes through but some is scattered by the particles in the material - Diffraction.
DNA cannot be crystallised, but in 1950 Maurice Wilkins developed a method of producing arrays of DNA molecules that were orderly enough for a diffraction pattern to be obtained, rather than random scattering.
Rosalind Franklin developed then a high resolution detector that produced very clear images of diffraction patterns from DNA.
Results: made deductions on the structure of DNA- helical shape, pitch of the helix, helix is 3.4nm apart.
Her observations were critically important in the discovery of the double helix structure of DNA by Crick and Watson.
Leading and lagging strands
The two ends of a strand of nucleotides in RNA or DNA are different - known as the 3’and 5’ ends.
The 3’ end in DNA has a deoxyribose to which the phosphate of another nucleotide could be linked - the phosphate would bond with the -OH group on the C3 of the deoxyribose.
The 5’ end in DNA has a phosphate that is attached to C5 of deoxyribose.
The direction of replication is always 5’ to 3’.
The two strands of DNA are antiparallel as run in opposite directions.
Because of the antiparallel structure of DNA the two strands have to be replicated in different ways.
On one strand DNA polymerases can move in the same direction as the replication fork so replication is continuous - leading strand.
On the other strand DNA polymerases move in the opposite direction to the replication fork so replication is discontinuous - lagging strand.
Prokaryotic DNA replication + enzymes used
Semi-conservative replication is carried out using many enzymes.
- DNA gyrase relieves the strain in the DNA molecule.
- Helicase unwinds the double helix and splits it into two separate strands. Single-stranded binding proteins keep the strands apart.
- DNA polymerase III adds nucleotides in a 5’ to 3’ direction. On the leading strand, it moves towards the replication fork.
- DNA primase adds a short length of RNA attached by base pairing to the template strand of DNA. This acts as a primer, allowing DNA polymerase to bind and begin replication.
- DNA polymerase III starts replication next to the primer and adds nucleotides in 5’ to 3’ direction - away from the replication fork on the lagging strand.
- Short lengths of DNA are formed between RNA primers on the lagging strand called Okazaki fragments.
- DNA polymerase I removes the RNA primer and replaces it with DNA. Leaves a nick in the sugar-phosphate backbone.
- DNA ligase seals up the nick.
Sanger sequencing
Frederick Sanger developed a method of basic sequencing widely used for 25 years.
Based on nucleotides of ddNA which contain dideoxyribose instead of deoxyribose - no OH group on carbon atom 3.
- If a dideoxynucleotide is at the end of a strand of DNA, there is no site to which another nucleotide can be added.
- In the sequencing machine single-stranded copies of the DNA being sequenced are mixed with DNA polymerase and normal DNA nucleotides, plus small nr of ddNA nucleotides.
- This replication is repeated 4 times with each base.
- The fragments of replicated DNA produced vary in length and are separated according to length using gel electrophoresis.
- Each band in the gel represents one length of DNA fragment produced by replication.
- There is only a band in one of the 4 tracks for each length of fragment - so the base sequence of DNA can be deduced easily.
Functions of DNA base sequences (coding and non-coding sequences)
- There are thousands of sequences of bases that code for proteins in the DNA of a species.
- These coding sequences are transcribed and translated when a cell requires a protein that they code for.
- Also, there are non-coding sequences, and some of them have important functions.
- Regulating gene expression - proteins can bind to them to either promote or repress the transcription of an adjacent gene.
- Introns - in many eukaryote genes the coding sequence is interrupted by one of more non-coding sequences. These introns are removed from mRNA before it is translated.
- Telomeres - repetitive base sequences at the ends of chromosomes. When the DNA of a eukaryote chromosome is replicated, the end of the molecule cannot be replicated, so a small section of the sequence is lost. Telomeres prevent parts of important genes at the ends from being lost during replication.
- Genes for tRNA and rRNA - transcription of these genes produces the transfer RNA used during translation and also the ribosomal RNA that forms much of the structure of the ribosome.
Bioinformatics
- Computers now allow huge amounts of data to be stored and analysed - a branch of biology called bioinformatics.
- Base sequences are the main type of data stored and analysed.
- Now, whole genomes can be analysed and sequenced.
- Used for: locating genes that code for polypeptides within genomes and searching for conserved sequences in the genomes of different organisms (for classification of living organisms).
Nucleosomes
DNA in eukaryotes is associated with proteins called nucleosomes.
Nucleosomes - globular structures that have 8 histone proteins with DNA wrapped around. Another histone protein called H1 binds the DNA to the core.
A short section of linker DNA connects the nucleosome to the next.
The 8 histones in the core have N-terminal tails that extend outwards - during condensation of chromosome in early stages of mitosis and meiosis, the tails of histones in adjacent nucleosomes link up and pull the nucleosomes together - supercoiling.
During interphase, changes to the nucleosomes allow chromosomes to decondense (uncoil) - the N-terminal tails are modified by adding acetyl or methyl groups - prevents adjacent nucleosomes from packing together.
The H1 histone protein is removed and the binding is loosened -allows DNA polymerase to carry out replication and transcription.
However, sometimes some sections of chromosomes remain condensed during interphase and genes in these sections are not transcribed - nucleosomes therefore help to regulate transcription in eukaryotes by controlling which sections of the chromosomes are condensed/decondensed during interphase.
Tandem repeats
- Within the genomes of humans and other species there are regions where adjacent sections of DNA have the same base sequence - tandem repeats.
- The length of the repeated sequence can be anything from 2 bases to 60 or more.
- Dimeric - two nucleotide repeat. Tetrameric - four base repeat.
- The nr of repeats varies between different individuals with some tandem repeats - variable nr tandem repeats.
- DNA profiling (fingerprinting) is based on variable nr of tandem repeats.
Stages in gene expression
- Gene expression - production of mRNA by transcription of a gene and then the production of polypeptides by translation of the mRNA.
- In prokaryotes - translation of mRNA immediately after transcription as no nuclear membrane.
- In eukaryotes - translation of mRNA in the cytoplasm after transcription in the nucleus.
Promoters and transcription
Promoter - control of gene expression involves this base sequence close to the start of a gene which is not transcribed and does not code for an amino acid sequence. Non-coding DNA with a function.
Transcription in prokaryotes:
- RNA polymerase binds directly to the promoter in prokaryotes and then starts transcribing.
- Repressor proteins can bind to the promoter and prevent transcription.
Transcription in eukaryotes:
- Proteins called transcription factors bind to the promoter allowing RNA polymerase to bind and start transcription. Repressor proteins can prevent transcription.
- RNA polymerase moves along the gene, assembling an RNA molecule one nucleotide at a time. In the 5’ to 3’ direction.
Identifying polysomes
Polysome - a group of ribosomes moving along the same mRNA as they simultaneously translate it.
Inheritance of acquired characteristics and epigenetics
- Lamarckism theory - acquired characteristics during the lifetime of a parent organism can be inherited by the offspring.
- Lamarckism was falsified.
- Discovery that DNA is the genetic material helped to falsify it - the environment of an individual during its lifetime cannot cause specific changes to the base sequences of their genes.
- Nevertheless, there is mounting evidence that the environment can indeed trigger heritable changes - epigenetics.
- Small chemical markers attached to DNA to fix the pattern of gene expression - these markers passed to daughter cells formed by mitosis and a small percentage persists and is inherited by offspring.
- Epigenome - the pattern of chemical markers established in the DNA of a cell.
- Epigenetics - research of the epigenome, methylation is one type of chemical marker.
Methylation and epigenetics
Cytosine in DNA can be converted to methylcytosine by the addition of a methyl group - catalysed by an enzyme and only happens where there is guanine on the 3’ side of the cytosine in the base sequence.
Methylation inhibits transcription - switches gene expression off for certain genes.
Environmental factors can influence the pattern of methylation and gene expression.
Fluorescent markers can be used to detect patterns of methylation in the chromosomes.
- Patterns are established during embryo development and reaches max at birth in humans and decreases during lifetime.
- At birth, identical twins have a very similar pattern of methylation but differences occur during their lifetimes (due to environmental factors).
Post-transcriptional modification
- Eukaryotic cells modify mRNA after transcription.
- Before mRNA exits the nucleus.
- In eukaryotes, coding sequence may be interrupted by one or more non-coding sequences (introns), which are removed from mRNA before translation.
- The remaining parts of the mRNA are exons - spliced together to form mature mRNA.
- Some genes have many exons and different combinations can be spliced together to make different proteins - increases the total nr of proteins an organism -can produce from its genes.
