Chromosomes, Genes, Alleles and Mutations

Eukaryotic chromosomes are made up of DNA and proteins.

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Gene: A heritable factor that controls a specific trait. It consists of a length of DNA occupying a specific locus on a chromosome. Genome: The genetic make-up/information of an organism. Allele: A specific form of a gene that differs from other forms of that gene by only a few bases, an occupies the same gene locus as other alleles for that gene. Gene Mutation A gene mutation is a change in the base sequence of a gene. It can occur as: A frame shift mutation o Addition o Deletion A substitution mutation

Sickle Cell Anaemia, an Example of Gene Mutation Sickle cell anaemia results from a mutation in the Hb gene which codes for haemoglobin. A substitution mutation occurs in the DNA sequence. The Thymine in the codon CTC is replaced with Thymine, causing the codon to become CAC. This results in an error in the mRNA strand during transcription as what would normally be GAG is replaced with GUG. This causes an error in translation because the anti-codon CTC, bearing Glutamic acid, is replaced with the anti-codon CAC, bearing Valine. The substitution of Glutamic acid for Valine results in the production of haemoglobin S instead of haemoglobin B, and this causes a change in the shape of the red blood cell. The red blood cells produced are sickle shaped instead of donut shaped and this affects their capacity to carry oxygen. However, sickle cell anaemia has its advantages as it gives resistance to malaria, so the mutation for sickle cell is common in areas with high instances of malaria.

Consequences of sickle cell anaemia include: Change in the shape of the bet-globin sub unit. Red blood cells become sickle shaped. The red blood cells live for 8 days as opposed to 80 days for normal red blood cells. Poor blood flow to peripheral tissues, and tiredness. Affects the red blood cells ability to absorb oxygen

Meiosis
nucleus.

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Meiosis is a reduction division that forms haploid nuclei from a diploid

Homologous Chromosomes are chromosomes of the same length, staining pattern, centromere position and posses genes coding for the same characteristics occupying the same locus. They do not necessarily have the same alleles for the genes. Meiosis A reduction division that results in the formation of four haploid cells from one diploid cell. Meiotic cells have an interphases similar to the one that occurs in mitosis. There is a G1, S and G2 phase Meiosis consists of two stages: Meiosis I and Meiosis II and involves a reduction division. Between Meiosis I and Meiosis II there is another brief interphase. Meiosis occurs in gametes and involves the formation of four haploid cells from one diploid cell. Meiosis involves sexual reproduction After Meiosis, each daughter cells is genetically different from the parent cell and from the other cells. In this sense it can be said to promote variation.

Meiosis I Interphase (specifically S phase) Chromosomes replicate to form two sister chromatids joined at the centromere. Prophase I Chromosomes condense. Nuclear envelope disintegrates. Homologous chromosomes pair up in a process called synapsis. The synaptonemal complex forms. o The synaptonemal complex is a protein that attaches homologous chromosomes to form a Tetrad.

Chiasmata are the points at which homologous chromosomes come into contact. Crossing over occurs, and the homologous chromosomes exchange genetic information. (This occurs at chiasmata) The centrosomes duplicate and they move away from each other. Spindle fibers begin to develop.

Metaphase I The Tetrad chromosomes line up at the metaphase plate. The spindle fibers attach to the chromosomes at the kinetochore. Anaphase I The Tetrad is split as the chromosomes move to opposite ends of the cell. Mendels law of independent assortment comes into play as the chromosomes are separated randomly.

Telophase I Cytokinesis occurs o Animals cleavage furrow o Plants cell plate After Meiosis I, two cells results. Each cell is genetically different from the parent and haploid.

Meiosis II Before meiosis II, there is no replication of genetic materials. Prophase II Nuclear envelope disintegrates. Chromosomes condense. Centrosome duplicates and begin to move away from each other. Spindle fibers begin to form.

Metaphase II Chromosomes migrate to the metaphase plate Spindle fibers attach to the sister chromatids at the kinetochore.

Anaphase II Sister chromatids are split pulled to opposite ends of the cell

Telophase II The nuclear envelope begins to reform. Spindle fibers disintegrate. Chromosomes uncoil to form chromatin. Cytokinesis occurs. The result of Meiosis II is four genetically unique haploid daughter cells.

Mitosis v. Meiosis Mitosis Result is two genetically identical, diploid daughter cells. Occurs in somatic cells. No synapsis of homologous chromosomes. In anaphase sister chromatids are pulled apart. No crossing over. Meiosis Result is four unique, haploid daughter cells. Occurs in gametes (germ cells). Synapsis of homologous chromosomes to form a tetrad. In anaphase I, homologous chromosomes are pulled apart. Crossing over occurs.

Chiasmata Formation The four sister chromatids become tightly linked in a process known as synapsis. A cut is made in the DNA of one chromatid, and another cut is made, in the same place, of a non-sister chromatid. The two non-sister chromatids join at the points where the cut was made and exchange genetic material (an exchange of paternal and maternal DNA). This is known as crossing over. After crossing over, the synaptonemal complex disintegrates but the sister chromatids remained joined at the point of genetic crossing over, and this forms an X shaped structure known as chiasmata. The chiasmata remain until late metaphase.

Meiosis and Genetic variation Two processes result in genetics variation in gametes: Crossing over o In meiosis homologous chromosomes pair up in a process called synapsis to form a tetrad o Chismata form between the homologous chromosomes and crossing over occurs o This allows for new allele combinations and thus variety! Random orientation of chromosomes in metaphase I

These two processes allow almost infinite genetic variation. Crossing over Crossing over results in genetic variation as it is an exchange of maternal and paternal DNA. It results in chromatids with new combinations of alleles. Chromatids with different combinations of alleles from either parent are known as recombinants (from the term genetic recombination). Chiasmata occur at random points along the chromosome, and more than one chismata can form between homologous pairs. Meiosis can form an almost infinite amount of genetic variation.

Random Orientation Random orientation of chromosomes during metaphase also contributes to genetic variation. Either maternal or paternal chromosomes can face either pole. The number of possible orientations is 2n where n is the number of chromosomes. In humans the number of possible orientations is 223.

Mendels Law of Independent Assortment Allele pairs separate independently, so the transmission of traits to gametes occur independent of each other. Mendels Law of Segregation

Maternal and paternal chromosomes separate and the gametes must have one allele for each pair. Mendels Law and Meiosis During metaphase the homologous pairs line up on the metaphase plate and orient randomly. This is known as random orientation. The chromosomes can end up at either pole (randomly). The way one homologous pair is facing does not affect the way the other homologous pairs are facing. This independent orientations forms the basis of Mendels law of Independent assortment. Unlinked genes are found on different chromosomes so when the chromosomes separate, they result in gametes with a random assortment of chromosomes and alleles.

Karyotyping

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A Karyotype is an ordered display of the chromosomes within an organism. In karyotyping, chromosomes are arranged according to their structure and homologous chromosomes are paired up. The factors used to identify and pair homologous chromosomes are: Size Banding pattern Centromere position Prenatal Diagnosis A karyotype of the foetus is carried out in order to identify genetic disorders. The karyotype is conducted with cells obtained through: Chorionic villus sampling Amniocentesis.

Reading a Karyotype Karyotypes can be used to determine gender or identify genetic abnormalities. In a karyotype, to determine gender you look at the X and Y chromosomes. If two X chromosomes are present, the individual is female, however if there is an X and a Y, the individual is male. The Y chromosome is easy to identify as it is much smaller than the X chromosome. Non-disjunction can also be identified by determining if the individual has an extra chromosome, or in some cases it missing a chromosome. Non-disjunction Non-disjunction results in a change in the number of chromosomes present in a gamete. This results in genetic abnormalities such a Downs syndrome. It occurs when sister chromatids of homologous chromosomes fail to separate either in anaphase I or anaphase II because moving apart is soooo hard. With Downs syndrome (trisomy 21), this results in an extra chromosome-21 as two copies of chromosome 21 are present in the gamete.

Fertilisation of the gamete results in 3 copies of chromosome-21. Individuals exhibit varying degrees of mental retardation. They are more susceptible to leukaemia, heart failure, Alzheimers and defects of the face, tongue and hands.

Theoretical Genetics
Genotype: the alleles of an organism Phenotype: the characteristics of an organism

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Dominant allele: an allele whose phenotype is expressed regardless of whether it is in the homozygous or heterozygous state Co-Dominant allele: A phenotypic situation in which two alleles both affect the phenotype in independent, distinguishable ways. Locus: The specific location of a gene on a chromosome Recessive allele: an allele whose phenotypes are only expressed when it is in the homozygous state. i.e. paired with an identical allele. Homozygous: Having two identical alleles of a gene. Heterozygous: Having two non-identical alleles of a gene. Carrier: A person who has one copy of a recessive allele that causes a genetic disease in the homozygous state. Test-cross: Testing a suspected heterozygote by crossing it with a known homozygous for that trait being investigated. A, B, O blood types A, B, O blood type is an example of multiple alleles and codominant alleles. Multiple alleles refer to genes that have three or more alleles. Human blood type has three alleles: o Ia o Ib o i a I and Ib are codominant, and i is recessive to both. Any two of these alleles are present in an individual. The possible genotypes and phenotypes are: Phenotype

Genotype

Ia Ia I I ii Ia Ib I i I i
b a b b

A B O AB A B

Sex and Genetics How Sex Chromosomes Control Gender The sex chromosomes, X and Y, are the 23rd chromosomes. Males can either have an X or a Y chromosome in their sperm, whereas females can only have an X chromosome in their egg. There is a 50% chance of the offspring being male, and a 50% chance of it being female. Males have the genotype XY and females have the genotype XX The Y chromosome (inherited from the father) has the SNR gene which initiates male determination.

Difference between the X and the Y The X chromosome contains some genes that cannot be found on the much shorter Y chromosome. The X and Y chromosome do share a short region which allows for pairing during meiosis. The X chromosome contains gene which are vital for life. This was discovered through gene mutations; OY is lethal, whereas OX (Turners syndrome) is not. The X chromosome contains 1,100 genes, whereas the Y chromosome contains 200 genes.

Sex Linkage It is the condition when genes responsible for a trait are found on the sex chromosomes. Examples Colour blindness o Female: XBXB, XBXb, XbXb o Male:= XBY, XbY o Where XB is normal vision

Haemophilia o Female: XHXH, XHXh, XhXh o Male = XHY, XhY o Where XH is normal blood type

Both traits are recessive and are found on the X chromosome. However, as males only have one X chromosome and the alleles for these conditions are not found on the Y chromosome, there are no male carriers only affected and unaffected males. However, a female can be homozygous or heterozygous for a recessive trait. Why females can be heterozygous for X-linked traits Females have two homologous X chromosomes Female carriers have two different alleles for the X linked recessive trait and thus are heterozygous for the trait.

Test crosses
Pedigree chart Female Male

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If the symbol is shaded, the individual is affected, if it is not then he or she is not. If it is half shaded, then the individual is a carrier. Traits are separated based on Mendels law of Independent assortment. Test-cross Monohybrid cross calculates the probability of the traits, in the offspring, of autosomal chromosomes at the same loci. (one particular trait, hence the word mono) Dihybrid cross calculates the probability of traits, in the offspring, of autosomal chromosomes for two traits, each at their own/respective loci. A di-hybrid cross is a cross between the F1 offspring of two organisms with two different characteristic. The characteristics are controlled by two different genes. We can use di-hybrid crosses to calculate the genotypic and phenotypic ratios of offspring involving unlinked autosomal genes. Difference between autosomes and sex chromosomes Sex chromosomes are the chromosomes that determine your gender. They are the X and Y chromosomes. Autosomes are the other chromosomes, which are not sex-chromosomes. There are 22 autosomes. In total there are 23 pairs of chromosomes: one pair of sex chromosomes and 22 pairs of autosomes. Linkage groups A pair of genes on a chromosome that are usually inherited together.

Crossing over results in exchange of alleles

Polygenic Inheritance Two or more genes acting on one phenotype o Hair o Skin colour

Polygenic inheritance in when more than one gene controls a characteristic. Usually there is co-dominance between these genes. Polygenic inheritance exhibits continuous variation Skin colour is controlled by four genes: A, B, C and D Each gene involved affects melanin production in the skin. Genes Genes are linked if they are located on the same chromosome E.g. Lathyrus Odoratus Linked genes are inherited together. Their ratio is non-Medelian (1:1:1:1) Any recombinants are a results of crossing over which occurs in prophase I.

Linked

Recombination The appearance of different gene combinations from the parents. The assortment of genes into different combinations from the parents. Chi-Squared A statistical test used to measure how much your data differs from the expected value. If the value is <5%, the difference is due to chance. If the value is >5%, then the difference is significant and you should probably go see a doctor because it might turn out to be something serious. Equation: X2 = sum(((O-E)2)/E) O = observed E = expected Degree of freedom = number of classes 1

Genetic Engineering and Biotechnology24/01/2012 02:42:00

Use of Polymerase Chain Reaction (PCR) to copy and amplify minute quantities of DNA PCR is used to copy and multiply small amount of DNA for manipulation or use. It is useful when a small amount of DNA is available but a large quantity is needed Process 1. Double stranded DNA is heated. This breaks the hydrogen bonds and separates the strand. 2. Primers are added so the polymerase can catalyse the synthesis of DNA. 3. PCR requires high temperature (so this occurs at a faster rate), thus a heat-resistant DNA polymerase enzyme, Taq polymerase (from Thermus aquaticus, a bacteria that live in hot springs i.e. a thermophilic bacteria) is used to create a double strand DNA molecule. Gel Electrophoresis Macromolecules DNA can be separated through gel electrophoresis. The molecules move in an electrical field and are separated based on size. The charge allows molecules to travel along the gel. It is used in DNA Profiling Details Two samples are obtained The samples of DNA are placed in a well (depression in the gel) The agarose gel is a sugar polysaccharide that forms a semi-solid matrix that separates DNA fragments based on size

An electrical current is placed on the two ends by placing electrodes at either end of the gel. The electrically charged molecules (phosphate groups) move to the cathode (positive end). DNA fragments, cut with restriction enzymes, move to the cathode. Samples can be compared to known values in order to be determined because the fragments are separated according to size, similar to chromatography.

Restriction Enzymes

Restriction enzymes are very specific, they cut in a specific way, at a specific place. DNA Profiling DNA profiling is a technique used to identify people by their DNA profiles. Each person has a unique DNA profile in that although sections of DNA (mainly the Extrons) such as the section that codes for Hemoglobin formation are identical for a majority of the human population, certain sections such as the satellite DNA (VNRTs found at the end of a DNA strand or SRTs found in the middle) are unique to every individual as a result of accumulated mutations. Essentially the difference in the Introns allows for DNA profiling. DNA profiling is done via gel electrophoresis. If the available amount of DNA to be profiled is minute, PCR (polymerase chain reaction) is carried out to copy and amplify the available amount because a large amount of DNA is required for the DNA to undergo testing. PCR is carried out at a high temperature of about 60 degrees Celsius to allow the process work

much faster. Heat is applied to break the hydrogen bonds between the strands of DNA, thereby causing the double helix to unwind, nucleotide bases are then added to the solution the DNA is in and Taq polymerase, an enzyme which can work at high temperatures is used to replicate the DNA. After the DNA has been copied via PCR, restriction enzymes are used to cut sections of the DNA. The ends formed by the cut DNA are either blunt or sticky depending on the type of restriction enzyme used. The fragments of DNA are then separated via Gel electrophoresis. In this process, the DNA macromolecules are separated in the gel according to size, by running a current through the gel. The DNA is placed in a gel, an electric current is placed at the two ends and the DNA fragments, which are charged, move to the positive end. This results in a pattern of banding on the Gel, similar to gel chromatography. These banding patterns are unlikely to be the same for two individuals and thus are used in DNA profiling. DNA profiling is a highly useful technique whose applications include paternity tests as the DNA profile of the father and the child would be similar, and forensic investigation as DNA profiling can be done from DNA samples obtained from blood, semen, tissues etc. It can also be used for taxonomy for classification of organisms as it can be used to determine genetic similarity.

How DNA is used in Forensic Investigations and Paternity tests Organisms have a short sequence of DNA which are repeated several times. These are called satellite DNA, and the lengths vary from person to person. These are used for profiling. A sample of the DNA is obtained. A reference sample is also obtained. The DNA sample is amplified via PCR The DNA is cut into fragments by restriction enzymes, The fragments are separated via gel electrophoresis based on the size of the fragments.

The bands are compared between the two samples, if the banding pattern is the same the DNA is from the same source If the patterns are similar the individuals may be related

Effects of mapping the human genome It is easier to study how genes influence human development It helps identify genetic diseases Gives more information on the origins, evolution and migration of humans. Allows the production of new drugs based on DNA base sequences of genes What happens when genes are transferred? When genes are transferred between species, the amino acid sequence of polypeptides remain the same because the genetic code is universal. The same four bases are used and interpretation of the codons remain the same. Basic Technique for Gene Transfer The gene of interest if cut out using a restriction enzyme, or reverse transcriptase is used to create a DNA copy from an mRNA strand. An extra guanine nucleotide is added to the end of the DNA strand to create a sticky end. The same restriction enzyme is used to cut plasmid DNA at a specific base sequences An extra cytosine nucleotide is added to the end of the plasmid to create a sticky end.

Genetic Modification
Uses of GM crops or Animals

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Examples of Golden rice: o Rice enriched with beta-carotene, which can be converted into Vitamin A in the body and can prevent blindness Factor IX producing sheep: o The sheep produces clotting factors (VII, IX or interleukins) in its milk. The factor can be extracted from the milk and used to treat haemophiliacs. Insecticide sweet corn: o Corn that is resistant to pests so the farmer does not have to spray potentially harmful insecticides.

Benefits Golden rice: o More specific breeding than with traditional methods. o Faster results obtained. o Ability to introduce characteristics from other species that would not naturally appear in the gene pool. o Increased productivity of the land (either animal or plant). Factor IX producing sheep o Using livestock to produce medicines o Make organisms more productive o Promote health and nutrition of animal/plant o Increase their growth and development Insecticide sweet corn o Increase resistance to diseases

Harmful Effects Genetically modified organisms and crops have many unknown interactions between the environment. May over compete with local species Reduces genetic variation May transfer the inserted gene to a weedy relative and create a super weed. Clones

 A group of genetically identical organisms A group of genetically identical cells derived from a single parent
Cloning allows you to produce multiple copies of the same genetic trait. How to make a clone for dummies Differentiated: (Dolly) o Treat females with GnRH to cause them to produce eggs o Remove nucleus from eggs using a micro-pipette o Culture nucleated cells. o Reduce the nutrient level so the cells stop dividing. o Fust the enucleated and nucleated cells together using a current of electricity o Culture cells until they become a morula (solid mass of cells) o Transfer several into the uterus. Dolly the sheep was cloned by taking udder cells from a donor sheep. These cells were cultured in a low nutrient environment so they would become dormant. An egg cell was taken from another sheep and the nucleus removed using a micropipette. The udder cells and egg cells were fused using a current of electricity. The fused cells developed like a normal zygote into an embryo. The embryo was planted into another sheep who acted as the surrogate mother. The lamb born (Dolly) was genetically identical to the donor sheep. Issues of Cloning Humans For Cloned embryos can be tested for genetic diseases Tissues do not suffer from immune incompatibility Regeneration research Against Cloned organs may show premature aging or cancer Religious reasons Embryos created and the rest will

be discarded May spin off into research in cancer and transplants Obtaining egg cells takes advantage of women.

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