How r u.

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Introduction to Molecular Biology(based on pages
25-36 of Kohane et als book)

• Chitta Baral
• Arizona State University

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Cells, genome, gene and DNA

• Almost all cells of a living organism contain an
  identical set of codes describing the genes and
  their regulation
• This code is encoded as one or more strands of
  DNA
• Cells from the different parts of an organism
  have the same DNA
• Distinction The portion of the DNA that is
  transcribed and translated into protein
• Genome entire complement of DNA molecules of
  each organism
• Overall function of genome Control the
  generation of molecules (mostly proteins) that
  will
• Regulate the metabolism of a cell and its
  response to the environment, and
• Provide structural integrity.

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Structure of DNA

• Made up of 4 different building blocks (so called
  nucleotide bases), each an almost planar
  nitrogenic organic compound
• Adenine (A)
• Thymine (T)
• Guanine (G)
• Cytosine (C)
• Base pairs (A -- T, C -- G)

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Structure of DNA -- 2

• Base pairs (A -- T,C -- G) are attached to a
  sugar phosphate backbone to form one of 2
  strands of a DNA molecule.
• Phosphate ((PO4) -3)
• Deoxyribose
• Two strands are bonded together by the base pairs
  (A T, C G).
• Results in mirror image or complementary strands,
  each is twisted (or helical), and when bonded
  they form a double helix.
• Direction of each strand (5 meaning beginning or
  3 meaning end of the strand)
• 5 and 3 refer to position of bases in relation
  to the sugar molecule in the DNA backbone.
• Are important reference points to navigate the
  genome.
• 2 complementary strands are oriented in opposite
  direction to each other.

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Structure of DNA -- 3
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Duplication of DNA

• Occurs through the coordinated action of many
  molecules, including
• DNA polymerases (synthesizing new DNA),
• DNA gyrases (unwinding the molecule), and
• DNA ligases (concatenating segments together)

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Transcription of DNA to RNA

• Why transcription
• (For genome) to direct or effect changes in the
  cytoplasm of the cell
• Need to generate new proteins to populate the
  cytosol (heteregenous intracellular soup of the
  cytoplasm)
• Note DNA is in the nucleus, while proteins are
  needed in the cytoplasm, where many of the cells
  functions are performed.
• Coding region of the DNA is copied to a more
  transient molecule called RNA
• Gene is a single segment of the coding region
  that is transcribed into RNA
• Generation of RNA from DNA (in the nucleus) is
  done trough a process called transcription

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Transcription

• RNA (Ribonucleic acid)
• Similar to DNA (except for a chemical
  modification of the sugar backbone)
• Instead of T contains U (Uracil) which binds with
  A.
• Is not double stranded but single stranded
• RNA molecules tend to fold back on themselves to
  make helical twisted and rigid segments.
• RNA is synthesized
• By unwinding the DNA double helix separating the
  2 strands.
• Using one of the strands as a template along
  which to build the RNA molecule
• Accomplished by Enzyme RNA polymerase (binds to
  promoter and copies or transcribes the gene in
  its full length)
• Resulting molecule is called Pre-mRNA
• Single stranded pre-mRNA is then processed.
• Splicing (mediated by spliceosome consisting of
  RNA and proteins) removes the introns.
• Ends modified (Capping modifies 5 end and
  Polyadenylation adds adenines at the 3 end) to
  enhance stability

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mRNA, ORFs, etc.

• Each cell has 20 to 30 pg of RNA (1 of the cell
  mass)
• The RNA that codes for proteins is called
  messenger RNA (mRNA)
• The part of DNA that provides that code is called
  Open Reading Frame (ORF)
• When read in the standard 5 to 3 direction, the
  portion of DNA before the ORF is considered
  upstream and the portion following the ORF is
  considered downstream.
• Promoter regions DNA sequence upstream of an ORF
• Specifically determine which gene to transcribe
• Transcription factors proteins that contain part
  that bind to specific promoter regions, thus
  activating or deactivating transcription of the
  downstream ORF

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Coding and non-coding RNA

• Not all RNA code for proteins
• 4 of total RNA is made of coding RNA
• Of the non-coding RNA
• Ribosonal RNA (rRNA) and transfer RNA(tRNA) are
  used in the various protein translational
  apparatus
• Small nuclear RNA (snRNA) found in eucaryotes,
  is part of the splicing apparatus
• Small nucleolar RNA (snoRNA) involved in
  methylation of rRNA
• Small cytoplasmic RNA (scRNA) plays a role in the
  expression of specific genes

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Prokaryotic and Eukaryotic cells

• Eukaryotes Organisms whose cells contain
  compartments or organelles within the cell, such
  as mitochondria and nucleus
• Animals, plants
• Prokaryotes Whose cells do not have these
  organelles (e.g. bacteria)
• Most prokaryotes have a smaller genome, typically
  contained in a single circular DNA molecule.
• Additional genetic information may be contained
  in smaller satellite pieces of DNA called plasmids

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More on transcription

• Most eukaryotic genes have exons (portions that
  will be put in the mRNA) and introns (that are
  normally spliced out)
• Some introns may have a promoter-like control of
  the transcription process
• If an intron is not spliced out then an
  alternative splicing product is created.
• Various tissue types can flexibly alter their
  gene products through alternative splicing
• Post-splicing (in Eukaryotes)
• The generated mRNA is exported (through nuclear
  pore complexes) to the cytoplasm
• In the cytoplasm, the ribosonal complex
  (containing hundreds of proteins and special
  function RNA molecules) acts to generate the
  protein on the basis of the mRNA code.

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Translation

• Process of generating a protein or polypeptide
  from an mRNA molecule is known as translation.
• Protein a polymer or chain of aminoacids, whose
  sequence is determined by the mRNA template
• 3 nulceotides code for 20 naturally occurring
  amino acids
• 43 64 thus several trinucleotide sequences
  (codons) correspond to a single amino acid.
• There is no nucleotide between codons, and a few
  codons represent start and stop.
• Notable exceptions code of naturally occurring
  selenocysteine is identical to that for a stop
  codon, except for a particular nucleotide
  sequence further downstream.

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Translocation of proteins

• A newly formed protein need to be translocated to
  the right place to perform its function (such as
  structural protein in the cytoskeleton, as a cell
  membrane receptor, as a hormone that is to be
  secreted by the cell, etc.)
• Signal peptide (header) part of the polypeptide
  that is one of the determinant of its location
  and handling

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Transcriptional programs

• Initiation of the transcription process can be
  caused by external events or by a programmed
  event within the cell.
• External events
• Piezoelectric forces generated in bones through
  walking can gradually stimulate osteoblastic and
  osteoclastic transcriptional activity to cause
  bone remodelling Heat shock
• Appearance or disappearance of new micro or
  macronutrients around the cell binding of
  distantly secreted hormones
• Internally programmed sequences of
  transcriptional expression (eg. clock and per
  genes)
• Pathological internal derangements of the cell
• Self-repair or damage detection programs can
  trigger apoptosis (self-destruction) under
  conditions such as irreparable DNA damage

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Biological function of proteins

• Enzyme catalysis DNA polymerases, lactate
  dehydrogenase, trypsin
• Transport hemoglobin, membrane transporters,
  serum albumin
• Storage ovalbumin, egg-white protein, ferritin
• Motion myosin, actin, tubulin, flagellar
  proteins
• Structural and mechanical support collagen,
  elastin, keratin, viral coat proteins
• Defense antibodies, complement factors, blood
  clotting factors, protease inhibitors
• Signal transduction receptors, ion channels,
  rhodopsin, G proteins, signalling cascade
  proteins
• Control of growth, differentiation and
  metabolism repressor proteins, growth factors,
  cytokines, bone morphogenic proteins, peptide
  hormones, cell adhesion proteins
• Toxins snake venoms, cholera toxin

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Gene expression studies

• Allow you to understand how a gene is regulated
  in a tissue or a cell type.
• Most useful way of studying gene expression is
  by measuring the levels of mRNA produced from a
  particular gene in a particular tissue.
• Application to understand certain biological
  process it is useful to study the differences in
  gene expression which occur during such
  processes. E.g.
• It is of interest to know which genes are induced
  or repressed, say in the liver, after a
  particular drug is taken.
• Or which genes are expressed in a tumor but not
  in the surrounding normal tissue.
• Some techniques for analyzing mRNA level of a
  single gene or to quantify gene expression
• Northern blots
• Quantitative reverse transcriptase PCR
  (QT-RT-PCR)
• DNA microarrays
• Proteomics (analysis of the protein synthesis
  that results from gene expression)

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DNA microarrays

• Consist of thousands of DNA probes corresponding
  to different genes arranged as an array.
• Each probe (sometimes consisting of a short
  sequences of synthetic DNA) is complementary to a
  different mRNA (or cDNA)
• mRNA isolated from a tissue or cell type is
  converted to fluoroscently labeled mRNA or cDNA
  and is used to hybridize the array.
• All expressed genes in the sample will bind to
  one probe of the array and generate a fluoroscent
  signal.
• A DNA microarray can interrogate the level of
  transcription of several thousand of different
  genes from one sample in one experiment. (One DNA
  microarray experiment reveals the mRNA levels of
  1000s of genes from one tissue or cell type at
  one time point)
• Particularly useful when studying the effect of
  environmental factors on gene expression.
• A fingernail size chip can interrogate 10,000
  different transcripts. Chip has 30-40 different
  probes half of them are designed to perfectly
  match 20 nucleotide stretches of the gene and the
  other half contains a mismatch as a control to
  test for specificity of the hybridization signal.

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SNPs (single nucleotide polymorphisms)

• Genetic basis for organismal diversity is due in
  large part to differences in sequences, also
  known as polymorphisms of each gene.
• Most of these polymorphisms differ from one
  another by one nucleotide and are known as SNPs.
• Due to the small portion of the genome coding for
  proteins and the redundancy in the mRNA code,
  only some SNPs will result in differently
  constructed proteins.
• It is believed that genomic markers such as SNPs
  spaced every 1000 bases will be sufficient to
  unambiguously resolve the span of genome
  associated with a phenotypic difference to a
  single gene.

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Gene clustering dogma

• Genes that appeared to be expressed in similar
  patterns are mechanistically related.
• I.e., if we can find genes whose expression
  patterns approximate one another we can possibly
  conclude that they have functions that are
  related.