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32771 32771 Presentation Transcript

  • Introduction To Molecular Biology By Salwa Hassan Teama (M.D)
  • Molecular Biology
    • Molecular biology ; the study of biology at the molecular level.
    • Molecular biology ; the study of gene structure and functions at the molecular level to understand the molecular basis of hereditary, genetic variation, and the expression patterns of genes.
    • The Molecular biology field overlaps with other areas, particularly genetics and biochemistry .
  • The Genome
    • The genome of an organism is the totality of genetic information and is encoded in the DNA (or, for some viruses, RNA ).
    commons . wikimedia . org / wiki / Image : Genome . jpg
  • Genome Database
    • The database is organized in six major organism groups :
    • Eukaryotes, Bacteria, Archaea, Viruses, Viroids and Plasmids.
    • All living things are grouped into three domain:
    • Eukaryotes;
    • Prokaryotes and
    • Archaea.
    Three Domain of Life
  • The Cell
    • The cell is the smallest living unit, the basic structural and functional unit of all living things. Some organisms, such as most bacteria , are unicellular (consist of a single cell). Other organisms, such as humans , are multicellular.
  • The Cell
    • Cells are stacked together to make up structures, tissues and organs. Most cells have got the same information and resources and the same basic material. Cells can take many shapes depending on their function.
    • Function of cells
    • Secretion (Produce enzymes).
    • Store sugars or fat.
    • Brain cells for memory and intelligence.
    • Muscle cells to contract.
    • Skin cell to perform a protective coating.
    • Defense, such as white blood cells.
  • Eukaryotic Cell
    • Eukaryotes are generally more advanced than prokaryotes . There are many unicellular organisms which are eukaryotic, but all cells in multicellular organisms are eukaryotic.
    • Eukaryotic cells are found in animals; plants; fungi and protists cell.
  • Eukaryotic Cell
    • Cell with a true nucleus , where the genetic material is surrounded by a membrane;
    • Eukaryotic genome is more complex than that of prokaryotes and distributed among multiple chromosomes;
    • Eukaryotic DNA is linear ;
    • Eukaryotic DNA is complexed with proteins called " histones;
    • Numerous membrane - bound organelles;
    • Complex internal structure;
    • Cell division by mitosis.
  • Prokaryotic Cell
    • Unicellular organisms, found in all environments . These include bacteria and archaea.
    • Without a nucleus ; no nuclear membrane ( genetic material dispersed throughout cytoplasm ;
    • No membrane - bound organelles ;
    • Cell contains only one circular DNA molecule contained in the cytoplasm;
    • DNA is naked (no histone);
    • Simple internal structure ; and
    • Cell division by simple binary fission .
  • Archaea
    • Archaea are prokaryotes; organisms without nucleus but some aspect of their molecular biology are more similar to those of eukaryotes.
  • Eukaryotic Cell Cycle
    • Eukaryotic Cell Cycle : defined as the sequence of events that occurs during the lifetime of a cell and is traditionally divided into four phases:
    • G1 = Growth and preparation of the chromosomes for replication
    • S = Synthesis of DNA
    • G2 = Preparation for mitosis
    • M = Mitosis
  • Central Dogma of Molecular Biology http://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookPROTSYn.html
    • The flow of genetic information as follows:
  • Deoxyribonucleic Acid (DNA)
    • Deoxyribonucleic Acid (DNA), the genetic material of all cellular organisms and most viruses, the gigantic molecule which is used to encode genetic information for all life on Earth.
  • Eukaryotic Cell
  • http :// genome . gsc . riken . go . jp / hgmis / graphics / slides / 01-0085jpg . html U . S . Department of Energy Human Genome Program, http :// www . ornl . gov / hgmis .
    • Thread like structure.
    • Located in the cell nucleus.
    • The storage place for all genetic information.
    • The number of chromosomes varies from one species to another.
    The Chromosome
    • In normal human cell DNA contained in the nucleus, arranged in 23 pairs of chromosomes ; 22 pairs of chromosomes (autosomes) ; the 23 chromosome pair determines the sex of individual and is composed of either two (x) chromosomes (female) or an (x) and (y) chromosome (male).
    The Chromosome
    • The basic units of inheritance; it is a segment within a very long strand of DNA with specific instruction for the production of one specific protein. Genes located on chromosome on it's place or locus .
    The Gene
    • DNA and RNA are long chain polymers of small compound called nucleotides. Each nucleotide is composed of a base ; sugar (ribose in RNA or deoxyribose in DNA ) and a phosphate group . The phosphate joins the sugars in a DNA or RNA chain through their 5` and 3` hydroxyl group by phosphodiester bonds.
    General Structure of Nucleic Acid
    • The structure of DNA was described by British Scientists Watson and Crick as long double helix shaped with its sugar phosphate backbone on the outside and its bases on inside; the two strand of helix run in opposite direction and are anti-parallel to each other. The DNA double helix is stabilized by hydrogen bonds between the bases.
    • This structure explains how genes engage in replication, carrying information and acquiring mutation.
    • The G+C content of a natural DNA can vary from 22-73% and this can have a strong effect on the physical properties of DNA , particularly its melting temperature.
    • There are four different types of nucleotides found in DNA , differing only in the nitrogenous base : A is for adenine ; G is for guanine; C is for cytosine and T is for thymine.
    • These bases are classified based on their chemical structures into two groups: adenine and guanine are double ringed structure termed purine , thymine and cytosine are single ring structures termed pyrimidine .
    • The bases pair in a specific way: Adenine A with thymine T (two hydrogen bonds) and guanine G with cytosine C (three hydrogen bonds).
    • Within the structure of DNA , the number of thymine is always equal to the number of adenine and the number of cytosine is always equal to guanine .
    • In contrast to DNA; RNA is a single stranded , the pyrimidine base uracil (U) replaces thymine and ribose sugar replaces deoxyribose.
  • Genomic DNA organization
    • Eukaryotic genes: DNA molecules complexed with other proteins especially basic proteins called histones , to form a substance known as chromatin . A human cell contains about 2 meters of DNA . DNA in body could stretch to the sun and back almost 100 times. So it is tightly packed.
  • Eukaryotic Chromatin
    • Eukaryotic chromatin is folded in several ways. The first order of folding involves structures called nucleosomes , which have a core of histones, around which the DNA winds ( four pairs of histones H2A, H2B,H3 and H4 in a wedge shaped disc, around it wrapped a stretch of 147 bp of DNA ).
  • DNA Forms
  • DNA Replication
    • DNA Replication: The DNA (all gene) duplication; the transfer the genetic information from a parent to a daughter cell ; the DNA base sequence are precisely copied.
    • Replication proceeds in a semiconservative manner, each strand of the DNA helix serves as a template for the synthesis of complementary DNA strands. This lead to the formation of two complete copies of the DNA molecule, each consisting of one strand derived from the parent DNA molecule and one newly synthesized complementary strand.
  • Mitochondrial DNA
    • Mitochondria is a membrane-enclosed organelle found in most eukaryotic cells .These organelles range from 1–10 micrometers ( μm ) in size.
    • Mitochondria generate most of the cell's supply of adenosine triphosphate (ATP).
    • Mitochondria are involved in a range of other processes, such as signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth .
    • Mitochondria have been implicated in several human diseases, including mental disorders ,cardiac dysfunction, [ and may play a role in the aging process.
    • Mitochondria has its own DNA.
  • Mitochondrial DNA
    • Mitochondrial DNA contains 37 genes, all of which are essential for normal mitochondrial function . Thirteen of these genes provide instructions for making enzymes involved in oxidative phosphorylation .
    • Oxidative phosphorylation is a process that uses oxygen and simple sugars to create adenosine triphosphate ( ATP ) , the cell's main energy source .
    • The remaining genes provide instructions for making molecules called transfer RNAs ( tRNAs ) and ribosomal RNAs ( rRNAs ) .
    • Mitochondrial genes are among the estimated 20,000 to 25,000 total genes in the human genome .
  • Function of The DNA
    • Deoxyribonucleic Acid ( DNA ), the gigantic molecule which is used to encode genetic information for all life on Earth.
    • The chemical basis of hereditary and genetic variation are related to DNA.
    • DNA directs the synthesis of RNA which in turn directs protein synthesis .
  • The Genetic Code
    • The purine and pyrmidine bases of the DNA molecule are the letters or alphabet of the genetic code. All information contained in DNA represented by four letters: A,T,C,G.
    • Three nucleotides of DNA (1st, 2nd and 3rd) form triplet codons. A group of codons constitute the genetic code, that can be translated into amino acid of proteins.
    • RNA Codon tRNA Amino Acids
  • The Genetic Code
    • The sequence of codons in the mRNA defines the primary structure of the final protein. Since there are 64 possible codons, most amino acids have more than one possible codon. Out of the 64 possible 3-base codons, 61 specify amino acids; the other three are stop signals (UAG, UAA, or UGA).
  • The RNA
    • Three major classes of RNA : messenger (mRNA), transfer (tRNA) and ribosomal (rRNA). Minor classes of RNA include small nuclear RNA ; small nucleolar RNA;………..
  • The RNA
    • The concentration of purine and pyrimidine bases do not necessarily equal one another in RNA because RNA is single stranded. However, the single strand of RNA is capable of folding back on itself like a hairpin and acquiring double strand structure.
  • Messenger RNA
    • mRNA molecules represent transcripts of structural genes that encode all the information necessary for the synthesis of a single type polypeptide of protein.
    • mRNA ; intermediate carrier of genetic information; deliver genetic information to the cytoplasm where protein synthesis take place.
    • The mRNA also contains regions that are not translated: in eukaryotes this includes the 5' untranslated region, 3' untranslated region, 5' capand poly-A tail.
  • Transfer RNA(tRNA)
    • All tRNAs share a common secondary structure represented by a coverleaf. They have four-paired stems defining three stem loops (the D loop, anticodon loop, and T loop) and the acceptor stem to which amino acids are added in the charging step.
    • RNA molecules that carry amino acids to the growing polypeptide.
  • Ribosomal RNA (rRNA)
    • Ribosomal RNA (rRNA) is the central component of the ribosome, the function of the rRNA is to provide a mechanism for decoding mRNA into amino acids and to interact with the tRNAs during translation by providing peptidyl transferase activity.
  • Ribosomes
    • Ribosomes ; Factory for protein synthesis; are composed of ribosomal RNA and ribosomal proteins (known as a Ribonucleoproteinor RNP). They translate messenger RNA (mRNA) to build polypeptide chains using amino acids delivered by transfer RNA (tRNA) .
  • Ribosomes
    • Eukaryotic ribosomes are larger. They consist of two subunits; a 60S subunit holds (three rRNAs 5S, 5.8S , 28S and about 40 proteins) and a 40S subunit contains (an18S rRNA and about 30 proteins) , which come together to form an 80S particle compared with prokaryotic 70S ribosome
    • Most mRNA are translated by more than one ribosome at a time; the result, a structure in which many ribosomes translate an mRNA in tandem, is called a polysomes.
    Polysomes
  • The Protein
    • Proteins are the basic building materials of a cell, made by cell itself; the final product of most genes.
    • Proteins are chain like polymers of a few or many thousands of amino acids. Amino acids are represented by codons, which are 3-nucleotide RNA sequences. Amino acids joined together by peptide bonds ( polypeptide) . Proteins can be composed of one or more polypeptide chains.
    • Proteins have many functions: provide structure that help cells integrity and shape (e.g. collagen in bone); serve as enzymes and hormones; bind and carry substance and control of activities of genes….
  • Four levels of a protein's structure:
    • Primary structure: Formed by joining the amino acid sequence into a polypeptide .
    • Secondary structure: Different conformation that can be taken by the polypeptide: alpha helix and strands of beta sheet.
    • Tertiary structure : Result from folding the secondary structure components of the polypeptide into three-dimensional configuration.
    • Quaternary structure : complex of several protein molecules or polypeptide chains, usually called protein subunits, which function as part of the larger assembly or protein complex.
  • Protein Structure
  • Gene Expression
    • Gene expression process by which a gene product (an RNA or polypeptide ) is made.
    • In transcription steps , RNA polymerase make a copy of information in the gene (complementary RNA) (mRNA) complementary to one strands of DNA.
    • In translation step , ribosomes read a messenger RNA and make protein according to its instruction. Thus any change in gene sequence may lead to change in the protein product.
  • Types of control in Eukaryotes
    • Transcriptional, prevent transcription, prevent mRNA from being synthesized.
    • Posttranscriptional, control mRNA after it has been produced.
    • Translational, prevent translation; involve protein factors needed for translation.
    • Posttranslational, after the protein has been produced.
  • Mutation
    • Mutation include both gross alteration of chromosome and more subtle alteration to specific gene sequence.
    • Gross chromosomal aberrations include: large deletions; addition and translocation (reciprocal and nonreciprocal).
    • Mutation in a gene's DNA sequence can alter the amino acid sequence of the protein encoded by the gene. Point mutations are the result of the substitution of a single base. Frame-shift mutations occur when the reading frame of the gene is shifted by addition or deletion of one or more bases.
  • Mutation
    • Mutations can have harmful, beneficial, neutral, or uncertain effects on health and may be inherited as autosomal dominant, autosomal recessive, or X - linked traits . Mutations that cause serious disability early in life are usually rare because of their adverse effect on life expectancy and reproduction .
  • Common Tools in Molecular Biology
    • Nucleic acid fractionation
    • Polymerase chain reaction
    • Probes, Hybridization
    • Vector, Molecular cloning
    • Nucleic acid enzymes
    • Microarray
    • DNA sequencing
    • Electrophoretic separation of nucleic acid
    • Detection of genes:
    • *DNA: Southern blotting; inSitu hybridization; FISH Technique
    • *RNA: Northern blotting
    • *Protein: Western blotting, immunohistochemistry
  • Human Genome Project
    • Goals
    • Identify all the approximately 20,000-25,000 genes in human DNA,
    • Determine the sequences of the 3 billion chemical base pairs that make up human DNA, store this information in databases,
    • Improve tools for data analysis, transfer related technologies to the private sector, and
    • Address the ethical, legal, and social issues (ELSI) that may arise from the project.
  • Molecular Biology : Uses
    • Various methods in molecular biology diagnose the different human diseases; diagnosis of an infectious agent, in malignancy, the presence of the genetic disease and in transplantation, paternity and forensic analysis.
    • The Most Recent Applied Technologies
    • Genetic engineering
    • DNA finger-printing in the social and forensic science.
    • Pre and postnatal diagnosis of inherited diseases.
    • Gene therapy.
    • Drug Design.
  • Molecular biology is facilitating research in many field including biochemistry, microbiology, immunology and genetics,…………………… Molecular biology allows the laboratory to be predictive in nature, it gives information that the patients may be at risk for disease (future).
  • Glossary
    • Alleles are forms of the same gene with small differences in their sequence of DNA bases.
    • Exon (Coding DNA): A gene sequence contains protein coding information.
    • Introns ( intervening sequence) (A noncoding DNA sequence ): Intervening stretches of DNA that separate exons.
    • Primary transcript: The initial production of gene transcription in the nucleus; an RNA containing copies of all exons and introns.
    • RNA gene or non-coding RNA gene : RNA molecule that is not translated into a protein. Noncoding RNA genes produce transcripts that exert their function without ever producing proteins. Non-coding RNA genes include transfer RNA (tRNA) and ribosomal RNA (rRNA), small RNAs such as snoRNAs, microRNAs, siRNAs and piRNAs and lastly long ncRNAs.
    • Enhancers and silencers: are DNA elements that stimulate or depress the transcription of associated genes; they rely on tissue specific binding proteins for their activities; sometimes a DNA elements can act either as an enhancer or silencer depending on what is bound to it.
    • Activators: Additional gene-specific transcription factors that can bind to enhancer and help in transcription activation .
    • Open reading frame (ORF) : A reading frame that is uninterrupted by translation stop codon (reading frame that contains a start codon and the subsequent translated region, but no stop codon).
    • Directionality: in molecular biology, refers to the end-to-end chemical orientation of a single strand of nucleic acid. The chemical convention of naming carbon atoms in the nucleotide sugar-ring numerically gives rise to a 5' end and a 3' end ( "five prime end" and "three prime end"). The relative positions of structures along a strand of nucleic acid, including genes, transcription factors, and polymerases are usually noted as being either upstream (towards the 5' end) or downstream (towards the 3' end).
    • 3' flanking region: Present adjacent to 3' end of the gene; often contain sequences which affect the formation of the 3` end of the message and may contain enhancers or protein binding sites.
    • 5' flanking region: A region adjacent to 5' end of the gene. It is not transcribed into RNA; it contains the promoter. May contain enhancers or other protein binding sites .
    • 3' untranslated region: The three prime untranslated region (3' UTR) is a particular section of messenger RNA (mRNA). It follows the coding region. It is a region of the DNA which is transcribed into mRNA and becomes the 3' end or the message, Several regulatory sequences are found in the 3' UTR. The 3' untranslated region may affect the translation efficiency of the mRNA or the stability of the mRNA. It also has sequences which are required for the addition of the poly(A) tail to the message (including one known as the "hexanucleotide", AAUAAA ).
    • 5' untranslated region: The five prime untranslated region ( 5' UTR ), also known as the leader sequence , is a particular section of messenger RNA (mRNA) and the DNA that codes for it. It is a region of a gene which is transcribed into mRNA. It starts at the site (where transcription begins) and ends just before the start codon (usually AUG) of the coding region. It usually contains a ribosome binding site (RBS), in bacteria also known as the Shine Dalgarno sequence (AGGAGGU). In prokaryotic mRNA the 5' UTR is normally short. Some viruses and cellular genes have unusual long structured 5' UTRs which may have roles in gene expression. Several regulatory sequences may be found in the 5' UTR .
    • Reverse Transcription: Some viruses (such as HIV, the cause of AIDS ), have the ability to transcribe RNA into DNA.
  • References & Online Further Reading
    • Robert F. Weaver. Molecular Biology. Fourth Edition. Page 600. McGraw-Hill International Edition. ISBN 978-0-07-110216-2
    • Innis,David H. Gelfand,John J. Sninsky PCR Applications: Protocols for Functional Genomics: ISBN:0123721865
    • Daniel H. Farkas. DNA Simplified: The Hitchhiker's Guide to DNA. Washington, DC: AACC Press, 1996, ISBN 0-915274-84-1.
    • William B. Coleman,Gregory J. Tsongalis: Molecular Diagnostics: For the Clinical Laboratorian: ISBN 1588293564...
    • Robert F. Mueller,Ian D. Young. Emery ' s Elements of Medical Genetics : ISBN. 044307125X
    • Daniel P. Stites ,Abba T. Terr. Basic Human Immunology: ISBN. 0838505430
    • Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter. Molecular Biology of the cell. ISBN. 9780815341055
    • http://www.pubmedcentral.nih.gov/
    • http://www.biomedcentral.com/1471-2105/2/8/abstract. Elena Rivas and Sean R Eddy Noncoding RNA gene detection using comparative sequence analysis
    • BMC Bioinformatics 2001, 2: 8doi:10.1186/1471-2105-2-8
    • www.medscape.com
    • http://www.medterms.com/script/main/art.asp?articlekey=4026
    • www.emedicine.com
    • www.ebi.ac.uk/2can good introduction to bioinformatics and molecular biology
    • http://www.genomicglossaries.com/
    • http://www.gene.ucl.ac.uk/nomenclature/guidelines.html defines the nomenclature for human genes
    • http://www.accessexcellence.org
    • http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/Codons.html
    • http://www.web-books.com/MoBio/
    • http://www.expasy.org
    • http://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookPROTSYn.html
    • Cell & Molecular Biology online: http://www.cellbio.com/recommend.html
    • http://www.ornl.gov/sci/techresources/Human_Genome/glossary/glossary.shtml%20
    • http://www.genome.gov/10000715
    • http://www.ncbi.nlm.nih.gov/About/primer/mapping.html
    • http://www.lilly.com/research/discovering/targets.html
    • http://www.informatics.jax.org/expression.shtml
    • www.wikipdia.com
    • http://www.biology.arizona.edu/cell_bio/tutorials/pev/page2.html
    • http://www.genome.ou.edu/protocol_book/protocol_index.html
  • Thank You