Biologie Moleculara in Endocrinologie

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Concepte Concepte fundamentale fundamentale , metode , metode şi tehnici de şi tehnici de biologie moleculară biologie moleculară în endocrinologie în endocrinologie Corin Badiu, 2006

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  • Concepte fundamentale, metode i tehnici de biologie molecular n endocrinologie Corin Badiu, 2006

  • Metode de biologie molecular n endocrinologieConcepte fundamentaleDe la clinica la cauza molecularaAnaliza expresiei genelorDetectarea mutaiilorMutageneza intitMsurarea transcripiei genelor i determinarea ARN-ului mesagerAnimale transgenice n studiul funciei genelor

  • Genomul: aspecte structurale i funcionale Secvenierea: date i consecine Proteom i transcriptomConcepte fundamentale

  • Aspecte structurale

  • From DNA to Chromosome organization

  • Dogma fundamentala a biologiei moleculareDNA to mRNA (transcription)mRNA to protein (translation)

  • GeneExon (codes for mRNA)

    Introns (spliced out during transcription)

    Promoters (part of the gene which binds to many transcription factor proteins that promotes transcription)

  • 53promoter regionexons (filled and unfilled boxed regions)introns (between exons)transcribed regiontranslated regionmRNA structure+1Gene structure

  • Structure of mRNA

  • Structure of tRNA

  • Translation

  • Genomul uman

  • Genomul umanStructura: 3,2 * 109 BP (23 cr) + 15.000 BP (mitocondrial) 3*104 gene: 100..10.000, (2000), < 2%Funcia:meninerea structurii cromozomiale (telomeri, matrice histonica); replicare celulara (centromer)Proteine (zona de gene structurale)Secvene reglatorii (promoter, inhibitor, izolator)Reglaj:Abunden a prot., timp, etap de dezvoltare, esutDiversitate:99,9% IDENTIC; 0,1% diferene, particulariti, susceptibilitate la boli

  • Genomul umanVariabilitate: polimorfism mononucleotidic (SNP), 1:1000 1,42 Mil. SNP, 60.000 n exoni (Science, 2001)Inserii, deleii, polimorfism repetitiv.Markeri:Polimorfism fr semnif. funcional.Boli monogenice f. rare - SAG, MEN, MODY,rezistena la androgeni Diversitate:Boli poligenice DZ, dislipidemii, Cvasc, cancer, boli infecioase: subsituii aa + F. risc

  • Genomul umanHuman Genome Project (1990 - 2001)Secvena: 3,2 Mild. BP 447 vol x 1000 pag x 1000 cuvLimbajul:Posibil 30.000 gene funcionaleSemne de carte: Sequence Taged Sites (STS)1 cM = 1 Mb, 1% recombinare 1996: MEN1,11q13, markeri transmii la bolnaviLocalizare:locus susceptibil, izolare ADN, identificarea genei, verificarea mutaiei la bolnaviAlgoritm: in silico screening pt cadru de citire, promoteri, secvene omologe cunoscute (1,5%)

  • Identification of genes - problemsGC rich islands Regions in some promoters that are much higher in GC content than surrounding DNA sequence Identify promoter region of many, but not all genes

    Pseudogenes Gene remnants that look like genes but are no longer active

    Genocopies More than one copy of an active gene, often with slight, but important, sequence variations

  • Identification of genes - problemsAbsence of co-linearity between genes and proteins

    Genomic DNA sequence generally does not mapdirectly onto codons for amino acids in proteins

    Failure of one gene one polypeptide dogma GenemRNAProtein

  • Consider genes with intronsnearly all protein-coding genes have intronsexceptions include histone genes

  • Translational frameshifting

    also yields more than one protein per mRNAmRNAAAA UAU GGC UUU

    AAA UAU UGG CUUlys tyr gly phelys tyr trp leuprotein

  • AplicaiiMEN

    MEN1: PTR Ad, Enteropancreatic, Pit ad, Adrenal, carcinoid11q13: 10 exoni, 1830 bp, 610 aa (menina) MEN1 mutant: n evaluare pt indicaiiMEN2: AD, (ret) MTC n 90%, Feocromocitom 50%, PTR 30%Exonii RET 10, 11, 13, 14, 15,16 Tiroidectomie profilacticClasa 3- risc maxim: 883, 918, 922 la 6 luniClasa 2- risc mare: 611, 618, 620, 634 la 5 aniClasa 1- risc mediu: 609, 768, 804, 891 10 aniClasa 0 risc mic: 790, 791 calcitonina periodic

  • Genomul umanMEN1www.ncbi.nlm.nih.govMEN1 + linksChandrasekharappa, Science 1997

  • Genetics


    Autosomal dominantly inherited Mutations of the MENIN-gene on Chr 11q13

    Parathyroid adenoma 95-100%Endocrine pancreatic tumours80%Pituitary tumours (Prolactin, GH) 30-50%Carcinoids (lungs, thymus, gastric, duodenal) 20-50%Lipomas 10-20%Thyroid Nodules 10-15%Lymphomas

  • Pedigree MEN-I

  • AplicaiiDefecte genetice ale axei de cretere

    Bermejo et al, TEM 11, 2, 2000

  • DNA microarray

  • Genomica funcional

  • Genomica funcionalGene: Determinarea tuturor secvenelor ce se exprimMecanismele de reglaj genetic, SNP ca factori predispozani, genetica populaionalProteine:Polimorfism cu semnificaie funcional: PROTEOM, TRANSCRIPTOM (esut, funcie). Produi diferii de degradare (POMC)Diversitate:Reglarea expresiei, specificitate tisularExperimental:oareci transgenici, knockout /knockdownDNA microarray, electroforeza 2D & Mass Spect

  • Diagnosticul genetic; screening genetic-Men1, SAG, DI central Terapie: tehnologia genic (insulina, rhGH, IGF1, rhPTH)terapia genic n neoplazii Medicina personalizatAplicaii

  • average base composition (G-C content) can be determined from the melting temperature of DNA Tm is dependent on the G-C content of the DNA

  • Types and rates of mutation

    Type Mechanism Frequency________ Genome chromosome10-2 per cell division mutation missegregation (e.g., aneuploidy)

    Chromosome chromosome6 X 10-4 per cell division mutation rearrangement (e.g., translocation)

    Gene base pair mutation10-10 per base pair per mutation (e.g., point mutation, cell division or or small deletion or 10-5 - 10-6 per locus per insertion generationMutation

  • Mutation rates* of selected genesGene New mutations per 106 gametes

    Achondroplasia 6to 40Aniridia 2.5to 5Duchenne muscular dystrophy 43to 105Hemophilia A 32to 57Hemophilia B 2 to 3Neurofibromatosis -1 44to 100Polycystic kidney disease 60to 120Retinoblastoma 5 to 12

    *mutation rates (mutations / locus / generation) can varyfrom 10-4 to 10-7 depending on gene size and whetherthere are hot spots for mutation (the frequency at mostloci is 10-5 to 10-6).

  • Polymorphisms exist in the genome

    the number of existing polymorphisms is ~1 per 500 bp there are ~5.8 million differences per haploid genome polymorphisms were caused by mutations

    New germline mutations

    each sperm contains ~100 new mutations a normal ejaculate has ~100 million sperm 100 X 100 million = 10 billion new mutations ~1 in 10 sperm carries a new deleterious mutation at a rate of production of ~8 X 107 sperm per day,a male will produce a sperm with a new mutationin the Duchenne muscular dystrophy geneapproximately every 10 seconds.

  • Types of base pair mutationsCATTCACCTGTACCAGTAAGTGGACATGGTCATGCACCTGTACCAGTACGTGGACATGGTCATCCACCTGTACCAGTAGGTGGACATGGTtransition (T-A to C-G)transversion (T-A to G-C)CATCACCTGTACCAGTAGTGGACATGGTdeletionCATGTCACCTGTACCAGTACAGTGGACATGGTinsertionbase pair substitutions transition: pyrimidine to pyrimidine transversion: pyrimidine to purinenormal sequencedeletions and insertions can involve one or more base pairs

  • Mutation is perpetuated by replication replication of C-G should give daughter strands each with C-G tautomer formation C during replication will result in mispairing and insertion of an improper A in one of the daughter strandsAC which could result in a C-G to T-A transition mutation in the next round of replication, or if improperly repairedCGCGandCGCGCAandCGTA

  • Mismatched (post-replication) repair53 the parental DNA strands are methylated on certain adenine bases mutations on the newly replicated strand are identified by scanning for mismatches prior to methylation of the newly replicated DNA the mutations are repaired by excision repair mechanisms after repair, the newly replicated strand is methylated

  • Infasurarea ADN

  • Recombinant DNA DNA from two different sources joined together.Cut the DNA and the plasmid using the same restriction enzyme (these enzymes recognize the same base sequences.Insert the foreign DNA into the plasmid.Replace the plasmid into the bacteriumAllow the bacterium to reproduce all future generations have the new DNACollect the product it might be insulin or growth hormone, or some other molecule.

  • PCRPolymerase Chain Reaction Used to amplifymake large amounts of a specific piece of DNA from a very small sample.Heat a starting quantity of DNA to separate the double helix.

    2.Add a collection of all four nucleotides, and DNA polymerase to copy the DNA, and some primers, and cool the sample.

  • PCR

  • Primers are short sections of DNA that are complementary to the region on both ends of the DNA that you wish to copy. Primers act as signals to tell DNA polymerase where to copy. As the solution cools, they stick to the DNA you wish to copy and allow polymerase to do its job.

    4.Heating the sample again unwinds the new duplicated strands; cooling again allows more primers to bind. If you repeat this as a cycle, you can make millions of copies of the original DNA.

  • Visualizing DNA Sequences

    A.So many bases, it is best to visualize them all in some organized fashion.1.Restriction enzymes can be used to cut the chromosomes from many cells into manageable pieces.

    There will be a collection of copies of fragment 1, which is a different size than fragment 2, and so on.

    3.The pieces can be ordered according to size using gel electrophoresis (moving the fragments in an electric field through a gel matrix). Larger pieces are more easily retarded by holes in the gel, so they travel less than smaller pieces: Figure 15.8

  • Sequence

  • Sequence

  • Analiza clonalitatii

  • Western Blot

  • Secventializare proteica

  • Secventializare proteica

  • Mutageneza tintita

  • DNA transformation: in vivo experiment

    Mice are injected either with Type R, non-virulentStreptococcus or with heat-killed, virulent Type S cells.The mice are healthy.Transgenic animals

  • X Mice are injected with both Type R, non-virulent andheat-killed, Type S Streptococcus DNA carrying genes fromthe virulent, heat-killed cellstransforms the non-virulentbacterial cells, making themlethal to the miceTransgenic animals

  • DNA transformation: in vitro experimentType R cellsType R coloniesType S cellsType S coloniesMixture ofType R and Type ScoloniesType R cells+ DNA fromType S cellsTransgenic animals

  • Transgenic animals

  • Plasmid DNA carrying thegrowth hormone geneInjected into nucleus of a fertilized mouse egg Egg implanted into uterusof surrogate mother mouse Mother mouse gives birth to transgenic mouseTransgenic animals

  • Mouse with growthhormone transgeneNormal mouseTransgenic animals

  • Mutation alters phenotype Phenotypic differences between individualsare due to differences between their genes These differences have arisen by mutation of DNAover many thousands of years

  • CloningTo make an exact genetic copy of; can be a gene, a cell, or an entire organism.

  • International Laboratory Directory

    ~600 Clinical and research laboratories ~1050 Inherited diseases

    ~700 clinical tests ~350 research only

  • Genetics in Specialty Care: Feature Search**Clinical laboratories

  • Genomica i ProteomicaEntrezGenomeMap viewerBLAST E-PCR VecScreen OMSSA

    This slide shows the structure of a typical human gene and its corresponding messenger RNA (mRNA). Mostgenes in the human genome are called "split genes" because they are composed of "exons" separated by"introns." The exons are the regions of genes that encode information that ends up in mRNA. The transcribedregion of a gene (double-ended arrow) starts at the +1 nucleotide at the 5' end of the first exon and includes allof the exons and introns (initiation of transcription is regulated by the promoter region of a gene, which isupstream of the +1 site). RNA processing (the subject of a another lecture) then removes the intron sequences,"splicing" together the exon sequences to produce the mature mRNA. The translated region of the mRNA (theregion that encodes the protein) is indicated in blue. Note that there are untranslated regions at the 5' and 3'ends of mRNAs that are encoded by exon sequence but are not directly translated.Hyperchromicity can be used to follow the denaturation of DNA as a function of increasing temperature. As thetemperature of a DNA solution gradually rises above 50 degrees C, the A-T regions will melt first giving rise to anincrease in the UV absorbance. As the temperature increases further, more of the DNA will becomesingle-stranded, further increasing the UV absorbance, until the DNA is fully denatured above 90 degrees C. Thetemperature at the mid-point of the melting curve is termed "melting temperature" and is abbreviated Tm. TheTm for a DNA depends on its average G+C content: the higher the G+C content, the higher the Tm. Note: G+Ccontent, G-C content, and GC content are equivalent terms.This slide shows the dependence of Tm on average G+C content of three different DNAs. Under the conditionsused in this experiment, E. coli DNA which has an average G+C content of about 50%, melted with a Tm of 69degrees C. The curve on the left represents a DNA with a lower G+C content and the curve on the rightrepresents a DNA with a higher G+C content. Tm is dependent on the ionic strength of the solution. At a fixedionic strength there is a linear relation between Tm and G+C content. For example, at 0.2M sodium ionconcentration, Tm = 69.3 + 0.41 (%G+C). Therefore, a DNA that is 50% G+C will melt at 89.8 degrees C in 0.2Msodium ion.This slide shows the three basic types of mutational events and their frequencies. We will be concentratingon "gene mutations," which are base pair mutations or small deletions or insertions.This slide illustrates the four basic types of base pair mutations. Two of them result in the conversion of onebase pair to another (base pair substitution). The others result in removal (deletion) or addition (insertion) ofone or more base pairs. (Note that a transition mutation results when a pyrimidine on one strand is convertedto another pyrimidine on the same strand. The complementary strand would see a conversion from one purineto the other purine.)The conversion of a C-G base pair to a T-A base pair takes two steps. If the tautomeric form of cytosine ispresent during DNA replication, an adenosine will be inserted into the daughter DNA strand (instead of thenormal guanosine). During the next round of DNA replication, the adenosine then serves as a template for theinsertion of a thymidine in the new DNA strand, resulting in a transition mutation (the conversion of a C-G to aT-A).For DNA to be repaired properly following the misincorporation of a nucleotide into the newly synthesizedDNA strand, the replication machinery must have a means by which to distinguish between the "old" (template)strand shown in black and the "new" (daughter) strand shown in red. After DNA replication takes place, thenewly synthesized DNA is methylated on certain adenine bases. This, however, does not occur right away -there is a "window of time" in which the newly synthesized DNA (in red) is not methylated. Thus, if a mutationoccurs in the new strand, the machinery can tell which is the template strand (presumable the correct strand)and the new strand (containing the mutation). It will then repair the mismatch by excision repair. Once sometime has passed, the new strand will also become methylated; at that point it will not be possible to distinguishthe correct nucleotide from the incorrect nucleotide at the site of a base pair mismatch.The DNA transformation experiments described in the next several slides show that DNA is the carrier of thegenetic information. The in vivo experiments show that something from the (heat-killed) virulent strain was ableto alter the (viable) non-virulent strain, converting some of the cells to virulent bacteria and killing the host. Wenow know that purified DNA confers this virulence.This in vitro experiment shows that purified DNA from Type S cells is able to be taken up by Type R bacteria. The process of getting functionally active DNA into cells is called DNA transformation. In this case,transformation by Type S DNA altered the "genotype" of the host cells, since new genes were introduced intothese cells thus altering their genetic constitution. The expression of this Type S DNA changed the "phenotype"of the transformed cells, making their colonies look "smooth" instead of "rough." Listen to audio descriptions of genotype* and phenotype* (requires RealPlayer).

    *Thanks to the NHGRI glossary of genetic terms.

    Transgenic experiments, which are usually carried out in mice, involve the transfer of a specific gene into thenucleus of a fertilized egg. The gene integrates randomly into the chromosomal DNA and can be engineered tobe expressed in every cell, or only in certain cells at certain times. In this experiment, introduction of thegrowth hormone gene into transgenic mice alters their genotype and confers the phenotype shown in the nextslide, which results from overexpression of growth hormone. Transgenic experiments show that specificphenotypic traits can be conferred by specific genes, and thus that DNA is the carrier of the genetic information. Listen to an audio description of transgenic* (requires RealPlayer).Other types of transgenic experiments involve mutation of specific genes in the mouse to determine thefunctions of those genes and to create mouse models of human genetic disease. The mutation of a gene in atransgenic mouse that eliminates the gene's function, is called a knockout mutation and the mouse carrying thatmutation is called a knockout mouse. Listen to audio descriptions of knockout* and mouse model* (requiresRealPlayer).

    *Thanks to the NHGRI glossary of genetic terms.

    The growth hormone transgenic mouse grows to a larger size than the normal, control mouse.Phenotypic differences between individuals are due in large measure to differences between genes. Evidence suggests that at least one-third of our genes are polymorphic, that is that there are differences in thenucleotide sequences in one-third of our genes when these genes are compared from one individual to anotherindividual. It is most likely that these differences occurred by mutation of DNA over many hundreds ofthousands of years of human evolution. It is also clear that new DNA mutations give rise to phenotypicdifferences between individuals, the most dramatic being those that give rise to genetic diseases. All of thisevidence indicates that DNA is the carrier of the genetic information.