* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Repetitive DNA info - A. Prokaryotes Eukaryotes Most codes for
Agarose gel electrophoresis wikipedia , lookup
Transcriptional regulation wikipedia , lookup
Maurice Wilkins wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
DNA barcoding wikipedia , lookup
Genome evolution wikipedia , lookup
Promoter (genetics) wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
DNA vaccination wikipedia , lookup
Transformation (genetics) wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Molecular cloning wikipedia , lookup
Endogenous retrovirus wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
DNA supercoil wikipedia , lookup
Point mutation wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Community fingerprinting wikipedia , lookup
Non-coding DNA wikipedia , lookup
Molecular evolution wikipedia , lookup
Repetitive DNA info A. Prokaryotes Most codes for proteins/tRNA/rRNA Coding seq. of nucleotides goes from start to finish-no interruption Eukaryotes Most (97%) does NOT encode protein / RNA. Most have functions unknown. Noncoding DNA consists of repetitive DNA –present in many copies in a genome usually not within genes and occur in different amounts in different species: a) tandemly repetitive DNA b) Interspersed repetitive DNA REPETITIVE DNA Reassociation Kinetics The existence of different classes of repetitive DNA is shown by reassociation kinetics. In this type of experiment genomic DNA is sheared into short lengths of about 1kb. It is then heated to denature or separate the two strands of the DNA. The solution is then allowed to cool and the amount of renatured double-stranded DNA measured at time points. To allow for different starting concentrations of DNA, reassociation time is usually represented by Co (starting concentration) X t1/2 (time taken for half the DNA to reassociate). 10-15% of typical eukaryotic DNA reassociates very rapidly. This DNA is highly repetitive and reassociates rapidly because there is a much greater chance of a single strand of DNA finding its complementary partner. This DNA is known as simple sequence DNA. Intermediate repeat DNA reassociates fairly rapidly but over a range of reassociation times which indicates a high degree of heterogeneity - this DNA is mainly composed of transposable elements. The slow component is DNA which occurs just once per haploid genome - single copy DNA. Simple Sequence DNA All eukaryotes have more than one type of simple sequence DNA. The repeat unit length is usually 5-10 bp but may be as short as 2 or as long as 200. These repeat units are arranged in head to tail tandem arrays which vary in length. The longest tandem array known so far is that of human alphoid DNA which has a total array length of approximately 5 million base pairs but certain simple sequences have an array length of only a few hundred base pairs. Most simple sequence DNA is found in heterochromatin - particularly at the centromere and at the ends or telomeres of chromosomes - though some simple sequences are scattered at different positions on different chromosomes. Tandem repeats and variable number tandem repeats (VNTRs) in DNA occur when a pattern of two or more nucleotides is repeated and the repetitions are directly adjacent to each other. An example would be: ATTCGATTCGATTCG in which the sequence ATTCG is repeated three times.Tandem repeats can be very useful in determining parentage. Short tandem repeats are used for certain genealogical DNA tests A variable number tandem repeats (VNTR) is a short nucleotide sequence ranging from 14 to 100 nucleotides long that is organized into clusters of tandem repeats, usually repeated in the range of between 4 and 40 times per occurrence. Clusters of such repeats are scattered on many chromosomes. Each variant is an allele and they are inherited codominantly. Coupled with Polymerase chain reactions, VNTRs have been very effective in forensic crime investigations. When VNTRs are cut out, on either side of the sequence, by restriction enzymes and the results are visualized with a gel electrophoresis, a pattern of bands unique to each individual is produced. The number of times that a sequence is repeated varies between different individuals and between maternal and paternal loci of an individual. The likelihood of two individuals having the same band pattern is extremely improbable. Southern blotting is also used to visualize the repeat numbers on the chromosomes. Once the tandem repeat has been found, identification of possible restriction sites on either side of the repeats are carried out. Using restriction enzymes will break the DNA into the repeat sequences plus a little on each end. The number of repeats will determine the length of the fragment of DNA. The repeat sequence itself can be used as a probe, or if the repeat is not long enough, a sequence from the upstream or downstream side can be used. The probe can either be radioactive or have a biotinylated linker for a fluorescent molecule. In looking at the VNTR data, two basic principles can be relied on: Tissue Matching- both VNTR bands must match. If the two samples are from the same individual, he must have exactly the same binding pattern. Inheritance Matching- the matching bands must follow the rules of inheritance. In matching an individual with his parents, a person must have one band that matches from each parent. If the relationship is further, such as a grandparent, then the matches must be consistent with the relatedness. Minisatellites are important because they are highly repetitive and dispersed into the genome. In humans, they are present in 60 autosomic loci and can be examined by digesting the DNA and hybriding with a monolocus probe or with another probe derived from a sequence that is common to each locus. Microsatellites or STR (short tandem repeats). Microsatellites have many uses: they can be used in forensics, genetic variability and parentage studies. Interspersed repetitive DNA is found in all eukaryotic genomes. These sequences propagate themselves by RNA mediated transposition and they have been called retroposons. Interspersed repetitive DNA elements allow new genes to evolve. They do this by uncoupling similar DNA sequences from gene conversion during meiosis. The recombinational events of meiosis create heteroduplexes composed of strands from each parental chromosome. These heteroduplexes lead to mismatch repair. The net result is the homogenization and elimination of sequence differences during meiosis. Gene conversion can be viewed as the force acting to create sequence identity within the gene pool of a species. This is a cohesive force acting to match up DNA sequences of individual organisms that comprise a species. In effect the gene conversion causes the DNA sequences to clump together within a species and by doing so creates the natural boundaries between species. The gene pool of a species consists of DNA sequences linked in a network by gene conversion events. An Alu sequence is a short stretch of DNA originally characterized by the action of the Alu restriction endonuclease. Alu sequences of different kinds occur in large numbers in primate genomes. In fact, Alu sequences are the most abundant mobile elements in the human genome The Alu endonuclease is so-named because it was isolated from Arthrobacter luteus. The Alu family is a family of polymorphisms in the Human genome. Alu sequences are about 300 base pairs long and are therefore classified as short interspersed nuclear elements (SINEs) amongst the class of repetitive DNA elements. There are over one million Alu sequences interspersed throughout the human genome, and it is estimated that about 10% of the mass of the human genome consists of Alu sequences. However less than 0.5% are polymorphic The shorter Alu or SINE repetitive DNA are specialized for uncoupling intrachromosomal gene conversion while the longer LINE repetitive DNA are specialized for uncoupling interchromosomal gene conversion. In both cases, the interspersed repeats block gene conversion by inserting regions of nonhomology within otherwise similar DNA sequences. The homogenizing forces linking DNA sequences are thereby broken and the DNA sequences are free to evolve independently. This leads to the creation of new genes and new species during evolution. By breaking the links that would otherwise overwrite novel DNA sequence variations, interspersed repeats catalyse evolution, allowing the new genes and new species to develop. Interspersed DNA elements catalyze the evolution of new genes DNA sequences are linked together in a gene pool by gene conversion events. Insertion of an interspersed DNA element breaks this linkage, allowing independent evolution of a new gene. The interspersed repeat is an isolating mechanism enabling new genes to evolve without interference from the progenitor gene. Because insertion of an interspersed repeat is a saltatory event the evolution of the new gene will also be saltatory. Because speciation ultimately depends on the creation of new genes, this naturally causes punctuated equilibria. Interspersed repeats are thus responsible for punctuated evolution and rapid modes of evolution. Microsatellites and human disease Microsatellites are also involved in a number of human genetic diseases which have been shown to be due to variations in the numbers of trinucleotide repeats. Fragile X syndrome is characterised cytologically by a break in the long arm of the X chromosome. Affected individuals suffer from mental retardation and it is one of the commonest causes of mental retardation. In early generations of a pedigree heterozygous carrier mothers pass the fragile X chromosome to 50% of their sons but it is rare for any of these sons to be affected by the disease - these phenotypically normal males are known as transmitter males. The daughters of these transmitter males however have a much greater chance of having affected sons. The gene associated with fragile X is known as FMR1. This gene has been shown to have a repeated CGG trinucleotide sequence in the first exon. In normal individuals this is repeated from 6 to 20 times. Individuals with 60 to 200 copies are unaffected female carriers or transmitter males. Individuals with more than about 230 copies are affected by the disease. The low number of repeats found in normal individuals is stable but the intermediate number of copies, found in carriers, is unstable and is said to be a premutation allele. When this allele passes through the male germ line there is no change but when passed through the female germ line there is an increase in the number of repeats. When the number of repeats exceeds around 230 expression of the FMR1 gene is affected, leading to the disease. Each time the unstable allele passes through the female germ line the number of repeats increases, leading to an increase in severity of the disease. This increasing severity of certain genetic diseases with succesive generations has been known for a long time and is termed anticipation. Other diseases which are associated with this type of trinucleotide repeat expansion include Huntington disease - where a CAG repeat expands, Myotonic muscular dystrophy - where a CTG repeat expands and Kennedy disease - where a CAG repeat expands. One explanation for the effect of these trinucleotide expansions in causing disease is that, above a certain length, they act as signals for conversion of chromatin into heterochromatin thus silencing the associated gene. It has been shown that the FMR1 gene becomes highly methylated in affected individuals. Methylation is associated with heterochromatin. DNA Fingerprinting Some simple sequence families form the basis for DNA fingerprinting. These families are called minisatellites. Minisatellites have a repeat unit length of 100 – 100,000bp and a total array length of 0.5 to 30 kb. The short array length is the reason they are called minisatellites to distinguish them from normal satellite DNA which has array lengths in the range of a few hundred kb. http://www.rvc.ac.uk/review/DNA_1/4_VNTRs.cfm Southern blot analysis of minisatellite array length variation forms the basis of DNA fingerprint analysis as it is currently practised. Tandem repeats are an array of consecutive repeats. They include three subclasses: satellites, minisatellites and microsatellites. The name "satellites" comes from their optical spectra. Illustration of satellite bands. By using buoyant density gradient centrifugation, DNA fragments with significantly different base compositions may be separated, and then monitored by the absorption spectra of ultraviolet light. The main band represents the bulk DNA, and the "satellite" bands originate from tandem repeats. Satellites The size of a satellite DNA ranges from 100 kb to over 1 Mb. In humans, a well known example is the alphoid DNA located at the centromere of all chromosomes. Its repeat unit is 171 bp and the repetitive region accounts for 3-5% of the DNA in each chromosome. Other satellites have a shorter repeat unit. Most satellites in humans or in other organisms are located at the centromere. Minisatellites The size of a minisatellite ranges from 100b to 100 kb. One type of minisatellites is called variable number of tandem repeats (VNTR). Its repeat unit ranges from 9 bp to 80 bp. They are located in non-coding regions. The number of repeats for a given minisatellite may differ between individuals. This feature is the basis of DNA fingerprinting. Another type of minisatellites is the telomere. In a human germ cell, the size of a telomere is about 15 kb. In an aging somatic cell, the telomere is shorter. The telomere contains tandemly repeated sequence GGGTTA. Microsatellites Microsatellites are also known as short tandem repeats (STR), because a repeat unit consists of only 10 to 100 bp and the whole repetitive region spans less than 150 bp. Similar to minisatellites, the number of repeats for a given microsatellite may differ between individuals. Therefore, microsatellites can also be used for DNA fingerprinting. In addition, both microsatellite and minisatellite patterns can provide information about paternity. The most famous case is President Thomas Jefferson and His Alleged Sons The term "polymorphism" describes the existence of different forms within a population, e.g., difference in the number of tandem repeats. All tandem repeat polymorphisms could result from DNA recombination during meiosis. Replication errors are the main source of mutations. It has been estimated that uncorrected replication errors occur with a frequency of 10-9 - 10-11 for each nucleotide added by DNA polymerases. Since a cell division requires synthesis of 6 X 109 nucleotides, the mutation rate is about one per cell division. A commonly observed replication error is the replication slippage, which occurs at the repetitive sequences when the new strand mispairs with the template strand. The microsatellite polymorphism is mainly caused by the replication slippage. If the mutation occurs in a coding region, it could produce abnormal proteins, leading to diseases. The Huntington's disease is a well known example. Figure 7-F-3. The mutation caused by replication slippage. In this figure, mispairing involves only one repeat. In fact, the slippage could cause several repeats to become unpaired. (a) Normal replication. (b) Backward slippage, resulting in the insertion mutation. (c) Forward slippage, resulting in the deletion mutation.