Download Repetitive DNA info - A. Prokaryotes Eukaryotes Most codes for

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.