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    Alu Sequences Can Be Mutagenic

    Alu sequences are repetitive sequences dispersed all over the genome andcan be within the genes or between the genes. Alu sequence is about 300base pairs, it is repeated randomly 300000 times all over the genome. Theydo not have physiological functions as the rest of the DNA, But sometimesAlu sequences can be mutagenic factors (they can cause genetic diseases if they cause a rearrangement to the chromosome), how?

    Cholesterol level is controlled by the endocytosis process of bloodcholesterol -which is carried on the LDL- by LDL receptors. When there issomething wrong with the LDL receptor, then cholesterol will stay in the

    blood and its concentration will increase. The condition in whichhypercholesterolemia is achieved is called familial hypercholesterolemia and this is because of defects in the LDL receptor which is responsible totake excess cholesterol by endocytosis and take it into the liver thatmetabolizes it. If there are defects in LDLreceptor then LDL cholesterol will stay inblood causing many cardiovasculardiseases.

    The figure represents the bloodlevel of cholesterol under differentphysiological conditions. The normalconcentration under normalphysiological condition ranges from 100to 250 or less mg/dl. A physiologicalcondition called heterozygosity is in

    relation to familial hypercholesterolemiawhich is in relation to inheritance of thedefect in LDL receptor gene.

    For each gene in somatic cellsthere are 2 copies; one copy is from the

    CHROMOSOME STRUCTURE & DNA REPLICATION

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    father and the other copy is from the mother. Regarding LDL receptor geneif there is one gene copy from one of the parents defected and the other isnormal then that inheritance causes heterozygosity, so the concentrationof cholesterol in the blood ranges from 300 to 500 mg/dl. If the two copiesare defected from the parents and inherited to the offspring then there ishomozygosity , so the LDL receptor is not functional at all and theconcentration of cholesterol in the blood ranges from 600 to 1000 or moremg/dl.

    How do Alu sequences cause the defect in the LDL receptor gene?

    The figure above represents LDL receptor gene, and the arrowsrepresent Alu sequences within this gene. Most of them are in the introns

    and two of them are in the exons. Because Alu sequences in LDL receptorgene are in close proximity to each other and they are homologous, there

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    will be unequal crossing over during meiosis which means there is amisalignment between the two chromosomes during the crossing over(exon 4 does not align with exon 4, and the same for Alu sequence andexon 5). The unequal crossing over results in the two products shown in theprevious figure. Deletion of exon 5 in one of the chromosomal products andduplication of it in the other cause a defect in the LDL receptor and causefamilial hypercholesterolemia.

    Chromosome Structure

    This is an electronmicroscopic

    picture of chromosome; wesee beats on astring repeated inan organized form(equal distancesbetween thesebeats). What is thestructure of thebeats?

    They are called nucleosomes which are found in eukaryotic genomes.It is very important to have nucleosomal structure for packaging of DNA onthe chromosomes. This is a magnificationof a nucleosome and it is composed of DNA and proteins (histones).

    The core of the nucleosome consistsof negatively supercoiled DNA of 146 basepairs long wrapped around 8 histoneprotein molecules (there are 2 moleculesof each type of these histone proteins

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    [H2A, H2B, H3, H4] forming an octamer). Thenucleosome as a whole is composed of DNA of 200 base pairs long wrapped around thehistone octamer, and monomer histonemolecule in the spacers that connectsnucleosomes with each other. These DNAmolecules wrapped around histones areresistant to nucleases, if you subject thechromosome to nuclease digestion and you run an electrophoresis for theDNA produced after nuclease digestion, what will you see in the picture of electrophoresis? We will see fragments of DNA and the length of afragment is 200 base pairs because nuclease will cut between nucleosomesand will be unable to digest the DNA which is wrapped around the histones,so we will see a ladder-like picture of fragments.

    Histones

    What is the significance of histones? Since the whole backbone of DNA isnegative and histones are positively charged so they stabilize the genomicstructure by these electrostatic interactions.

    The sequences of the different types of histones are homologous andthey are homologous also in every species all over the evolutionary tree.Why are there different types of histones? They might have differentamounts of positive charges that do more stabilization for the DNA on thechromosome. Also there will be different responses of these histones tomodification.

    General information about histones:

    1) Histones are small proteins; each histone gene has one exon andno introns.

    2) They are positively charged because they are rich in arginine andlysine (positively charged amino acids because they are basic, andthey are basic because they have a basic group with very high pk a

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    [12 for Arg R group and about 10.5 for Lys R group], they will notbe titrated at physiological pH so they stay positively charged).

    3) They also interact with negatively charged DNA.4) They can be extensively modified.

    Modification of Histones

    Modification means addition or removal of groups. It is one of theimportant mechanisms to regulate gene expression. When a gene beingexpressed, the DNA must unfold, the wrapped DNA around histones mustbe unfolded and must be exposed so no histones will stay in a gene or in aDNA when it is expressed. When it is not expressed all of them arepackaged with each other. By receiving the signal for expression everythingwill be unfolded and the chromosome or the DNA will be exposed withoutany nucleosome structures on it for other proteins to come and activate itsexpression. So how will DNA be unfolded from the nucleosomal structures?By modification to delete the positive charges of histones, then there willbe uncoiling or unfolding of DNA to be ready for expression.

    There are different processes of modification of histones:

    1) Phosphorylation of histones gives negative charge. In whichamino acid residue of histones? Serine or Tyrosine that have thehydroxyl group in R groups.

    2) Poly (ADP) Ribosylation .3) Methylation : methyl groups on some reactive groups to mask

    them and that affects their function. 4) Acetylation : Adding an acetyl group to affect active group in the

    histone.

    Hypoacetylation : There is an increase in the positive charge of histones that leads to increase the hold of histones to DNA soDNA is not exposed to proteins that cause expression.Hypoacetylation is associated with repression of geneexpression and transcription. Hypoacetylation is caused by the

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    enzyme histone deacetylase which is activated byretinoblastoma protein (RB).

    Hyperacetylation : to mask positive charges on histones sothere will be destabilization of the nucleosomal structures andthe DNA will be unfolded and hyperacetylated.Hyperacetylation is associated with activation of transcriptionand gene expression. Hyperacetylation is activated by sometranscriptional factors.

    Telomeres

    The ends of chromosomes are called telomeres . They are DNA sequencesrich in G and C bases. A telomere is a satellite repeated sequence andranges from 1 Kb to more than 12 Kb.

    They are important since they are considered as caps to protect theends of the chromosome from shortening and from karyotypicrearrangements during DNA replication.

    The young cell has a very long telomere while the aged cell has ashort telomere. During replication always there is a loss of DNA sequencesfrom the ends of the chromosome and this is why in aged cells there are nomore telomeres. Genes will be also degraded from the ends of thechromosomes and that will cause aging and death of those cells. So one of

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    the reasons of aging is degradation of telomeres causing no moretelomeres to protect our chromosomes from degradation during DNAreplication.

    What causes telomeres always in young cells to have full-length of sequences? An enzyme called telomerase . So every round of DNAreplication, telomerase works on telomeres and extends them to theproper length, with age telomerase activity decreases. What is the reasonthat cancer cells are not aged and they are always active and do not die?Because they have very active telomerase enzyme that always works ontelomere and increases the length of it.

    Packaging of DNA into chromosomes

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    The previous figures represent packaging of DNA into chromosomes. Inorder to accommodate the very large DNA molecule in the very smallnucleus there must be a mechanism of packaging of DNA into chromosome.

    The Doctor played a video about DNA packaging (link:http://www.youtube.com/watch?v=gbSIBhFwQ4s ).

    DNA passes through many steps of packaging, it starts as naked DNAthen it forms the nucleosomal structure, then these nucleosomes fold oneach other forming structures that refold on each other. There is anincrease in the thickness (diameter) through the steps of packaging (there isan increase in the diameter of the helix related to the diameter of thestructure with nucleosomes).

    After refolding of nucleosomes with each other, the foldednucleosomes bind to a protein called scaffold protein. Many scaffoldstructures after that step refold on each other to finally form the chromatidor the chromosome structure.

    DNA Replication

    http://www.youtube.com/watch?v=gbSIBhFwQ4shttp://www.youtube.com/watch?v=gbSIBhFwQ4shttp://www.youtube.com/watch?v=gbSIBhFwQ4s
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    This is the mammalian cell cycle, in S phase DNA synthesis or DNAreplication take place and histones are synthesized (after G1 phase). In G1phase there is a rapid growth and preparation for DNA synthesis (DNAsynthesis requires a lot of proteins and enzymes) so in this phase there issynthesis of the required proteins and enzymes for DNA replication. Beforecell division DNA must be replicated.

    DNA replication is semiconservative. What does it mean?DNA is double-stranded, it isimportant to be adouble-stranded

    structure for DNAreplication becauseeach strand will actas a template forthe synthesis of the new moleculesof double-strandedDNA that resemblethe parental DNAmolecule, if DNA was not double-stranded then that goal would not beachieved. As you see in the figure when the double-stranded DNAreplicates, the two strands must unfold and each strand acts as a template,and that will continue till you reach the end of the DNA molecule.

    So each daughter (new) DNA molecule has the original parental DNAstrand as a template and a newly synthesized strand which is

    complementary and antiparallel to the parental DNA template. Andbecause of this the two daughter DNA molecules exactly resemble theparental DNA molecule, so the DNA replication is semiconservative .

    Our DNA is very big and requires hours to finish its replication. And toreplicate in few hours there must be many sites where the chromosome

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    starts the replication from (there is no one single site to initiate thereplication on each chromosome). Every site at which the replication startsfrom is called the origin of replication . Prokaryotes have only one origin of replication because they have small genome.

    Our genome has multiple origins of replication in order to replicatethe entire DNA in few hours (if there was one origin of replication, it wouldtake years to finish DNA replication).

    On each origin of replication there is melting of the double-strandedDNA into single-stranded and the replication starts bidirectionally to formreplication folds, then these replication folds diffuse with each other onthe chromosome and finish the replication in the required time.

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    LECTURE OF 3 OCTOBER

    To start DNA replication the nucleosomes must be removed byhyperacetylation through signals to make DNA exposed to proteins. Amongthese proteins is a protein called dnaA protein which binds to the origin of replication and causes melting of DNA. When does DNA melt? After dnaAproteins molecules aggregate with each other. Then dnaB and dnaC proteins bind into the melted DNA region in the single-stranded DNAmolecule. dnaB protein helps the DNA to unwind in order for the comingproteins to reach DNA templates and continue replication.

    Done by:

    Amjad Habeb