1. Science 101 Jackie Wirz, PhD Sylvia Nelsen, PhD

2. Science 101* * This SLIDESHARE Presentation has been modified from the July 22nd 2011 OHSLA CE Presentation. Some slides have been deleted or condensed for brevity (a little bit, at least – this presentation is still LONG).

3. Science 101* Since a bunch of slides are different than a live presentation (shocking, I know!), I’ve attempted to bridge the gap by providing some text in yellow boxes to the slides. This provides a quick orientation to what’s going on; however, as always, if you have any questions please feel free to contact us directly.

4. For the most part, the Tweets are meant to be humorous. They also signify the end of a section. Lesson in a Tweet: Nerds of the world, unite #OHSLA

5. organization & objectives

6. Agenda

8. Cells & the Central Dogma

9. Molecular Architecture of the Cell

10. The Care & Feeding of DNA

12. Small molecules

13. Big molecules (A&P)

14. Translational Medicine

17. Learning Objectives

18. Cell Biology Major Organelle Replication, Repair Transcription Translation

19. Major Molecules DNA RNA Protein Others (sugars, lipids)

20. Genetics Genes and their inheritance

21. The Big Picture Human Disease & Databases

22. Science …with a capitol “S”!

23. As entertaining as this cartoon is, the bottom line is that we cannot summarize science into 140 characters or less… Or in a 4 hour CE Class. However, we can try to give you enough information to provide you with the essential vocabulary necessary to orient yourself in the biomedical sciences. Image © Jorge Cham, PhD Comics www.phdcomics.com

24. what are we talking about?

25. Science (from Latin: scientia meaning "knowledge") is a systematic enterprise that builds and organizes knowledge in the form of testable explanations and predictions about the world.

26. Science can be classified into many different categories.

27. A humorous look at how different scientific disciplines view each other. Image from xkcd.com

28. If we alter the graphic to focus on the biomedical health sciences, many disciplines can be added... Biomedical Health Sciences DEVELOPMENTAL BIOLOGY INFORMATICS GENETICIST MD CELL & MOLECULAR BIOLOGY IMMUNOLOGY BIOMEDICAL ENGINEERS NEUROSCIENCE Image from xkcd.com

29. Modern science promotes collaboration. Some natural associations are MDs working with geneticists, and biologists working with cell & molecular scientists. Biomedical Health Sciences DEVELOPMENTAL BIOLOGY INFORMATICS GENETICIST MD CELL & MOLECULAR BIOLOGY IMMUNOLOGY BIOMEDICAL ENGINEERS NEUROSCIENCE Image from xkcd.com

30. However, modern science is truly a collaborative endeavor! Biomedical Health Sciences DEVELOPMENTAL BIOLOGY INFORMATICS GENETICIST MD CELL & MOLECULAR BIOLOGY IMMUNOLOGY BIOMEDICAL ENGINEERS NEUROSCIENCE Image from xkcd.com

31. Truth be told, we are all scientists, seeking to build and organize information. SCIENTISTS Image from xkcd.com

32. The commonality is that we all speak geek. We aim in this course to provide you with some of the fundamental vocabulary to understand biomedical geek. SCIENTISTS speak geek

33. Lesson in a Tweet: Science… The Anti-Romance Language #truism

34. essential cell biology

35. cells & the central dogma molecular architecture of the cell on the care and feeding of your DNA transcription and translation

36. cells & the central dogma

37. Biomedical science spans an impressive range of size. From the observable (mm) to the atomic (nm), scientific understanding is, in part, coupled to our ability to “see” molecules, compounds and humans with an analytical eye.

38. In 1665, Robert Hooke published Micrographia, a book that described his microscopic observations and which is notable for the first use of the word “cell” as understood by biologists. Below is a drawing that depicts the cellular structure that remains in cork.

39. Below is a drawing created in 1880 depicting a living plant cell that divided over the course of 2.5 hours. The second half of the panel shows a similar cell photographed under a modern light microscope.

40. Cells come in all shapes and sizes! images from CIML immunology, Nuvo Manufacturing and Wikipedia

41. Cells are either Eukaryotic or Prokaryotic. Most cells aren’t this pink… Eukaryotic cells contain organelle. Image from Genomes, 2nd ed

42. Image from Words and Unwords c/o Zazzle.com

43. Prokaryotic cells can be quite diverse, and are capable of living in extremely harsh conditions. Images from Society for General Microbiology and Phototake

44. E. coli is a famous prokaryote. In the image below, the white areas inside the cell are concentrated areas of DNA.

45. This beautiful watercolor painting was painted by David Goodsell. It presents an E. coli in a way that underscores the complexity of a prokaryotic cell. The area boxed in white is shown close up below. Image from David Goodsell (mgl.scripps.edu/people/goodsell/)

46. Cells are either Eukaryotic or Prokaryotic... Let’s check out the characteristics of a eukaryotic cell. Eukaryotic cells contain organelle. Image from Genomes, 2nded

47. Image from Skreened.com

48. This is a schematic of the major parts of a eukaryotic cell: major organelle, the cytosol and the plasma membrane.

49. The nucleus houses our DNA, and is enclosed by the nuclear envelope. The envelope is porous, to allow for entry and exit of proteins and small molecules involved with DNA replication, repair, transcription, etc. Also note, DNA is only found condensed into chromosomes at very specific times during cell division.

50. Fun fact: brown fat contains a high number of mitochondria; brown fat generates body heat in newborn or hibernating mammals. Mitochondria are the powerhouses of the cell – most of the cell’s supply of ATP and other molecules involved with energy and cellular metabolism. Mitochondria are also important for the storage of calcium, regulation of programmed cell death (apoptosis), and other cellular functions.

51. The endoplasmic reticulum is a “lacey” system of membrane networks that can be divided into three major groups. 1) The rough endoplasmic reticlum (rER) synthesizes proteins. 2) The smooth ER synthesizes lipids and steroids while & regulats calcium concentration. It is also involved with the metabolism of carbohydrates, steroids, and other small molecules. 3) the sacroplasmicreticula regulates calcium levels.

52. The Golgi apparatus processes and packages macromolecules (like proteins and lipids). Essentially, the Golgi serves as a mail service that tags and directs proteins. This is essential for proteins that are secreted outside of the cell. Processing of macromolecules includes addition of carbohydrates (glycosylation) or phosphates (phosphorylation): these are post-translational modifications.

53. The cytoskeleton is comprised of a variety of proteins that create a scaffold within the cytoplasm. The filaments can be divided into three main groups, that differ in the structure and function within the cell. Image from Tobin/Dusheck Asking About Life 2/3

54. The cytoskeleton is especially beautiful when fluorescently tagged (images are examples of fluorescent microscopy) The cytoskeleton is most easily visualized during cell division, when microtubules are easily seen as they help to separate chromosomes

55. Membranes enclose all organelle. The plasma membrane encloses the entire cell. Membranes are selectively permeable to molecules, are composed of phospholipidbilayers and are embedded with proteins and other molecules.

56. Lysosomesbreak down waste and cellular debris. Their interior is acidic, which is necessary for its enzymes to function. Peroxisomesbreakdown fatty acids, D-amino acids and other chemicals in addition to synthesizing certain chemicals necessary for function of the brain and lungs. Additional functions for peroxisomes have been proposed, but experimental evidence is strongly debated.

57. As beautiful as the previous cartoons are, they fail to adequately depict how crowded a eukaryotic cell is! This electron micrograph clearly demonstrates the tight packing of a cell.

58. Lesson in a Tweet: What’s the favorite form of communication for molecular biologists? A cell phone!

59. the central dogma

60. DNA is transcribed into RNA which is translated into PROTEIN.

61. We are now going to focus on the major molecules of the cell: DNA, RNA and Protein.

62. Lesson in a Tweet: Dogma. More than a movie.

63. molecular architecture of macromolecules

64. DNA

65. DNA (deoxyribonucleic acid) is composed of a sugar-phosphate background to which a base is added. The resulting molecue is a nucleotide. Guanine, Cytosine, Adenine and Thymine are the four natural bases in DNA. DNA has a directionality, which is defined as having a 5’ (five prime) or 3’ end. DNA sequences are generally read 5’ to 3’

66. The double strand DNA (dsDNA) forms a specific structre known as the double helix. This is a schematic that highlights the specific turns that characterize the double helix. DNA naturally base pairs: A with T, G with C. This process is extremely efficient and reproducible. The process of matching two complementary strands of DNA is known as hybridization.

67. Image from Wikipedia.org c/o Benjah-bmm27 This is a space filling model of a DNA double helix. The phosphate backbone is clearly visible as orange & red knobs on the exterior edge of the helix.

68. Hybridization is a non-covalent, highly specific interaction between molecules. In DNA, this means that a double helix can be denatured (unfolded) using high temeprature of high pH creating single stranded DNA (ssDNA); after cooling or lowering the pH, the double helix will come back together in the original conformation. Hybridization is specific, efficient and the backbone of many DNA techniques such as FISH and PCR.

69. DNA is organized into genes, which are functional units of heredity. Genes have associated regulatory regions and transcribed regions/functional sequence regions. start site stop site gene of interest template strand promoter terminator

70. Eukarotic genes have introns and exons. Introns are regions of DNA that do not code for protein, and are spliced out of the mRNA. Once the introns have been removed, exons are exported out of the nucleus to begin translation. introns exons

71. Genes code for different proteins. There are ~22,000 genes in the human body.

72. Genes can be oriented on DNA in a variety of directions. The arrows depicted below follow the template strand, such that the coding strand (and the resulting transcribed mRNA ) always goes from 5’ to 3’.

73. The larger image depicts the DNA from 0.1% of one chromosome that has been released from its storage proteins. The inset pictures shows condensed chromatids. Image from Berkeley Open Computing Facility

74. DNA is packaged into a smaller volume to fit into the cell. DNA wraps around protein complexes (histones) to create chromatin. Chromatin can be further packed into chromosomes during specific times in the cell cycle.

76. RNA

77. RNA, ribonucleic acid, is very similar to DNA. It is formed out of a phosphate and sugar backbone to which four different bases are added to create nucleotides. The sugar in RNA is ribose (DNA uses deoxyribose): the RNA bases are adenine, cytosine, guanine or uracil. A bases pairs with U, C with G.

78. DNA: dideoxyribonucleic acid sugar = deoxyribose bases = CATG C A T G RNA: ribonucleic acid sugar = ribose bases = CAUG C A U G

80. RNA was originally thought to just be the intermediate molecule between DNA and protein. However, RNA has been shown to have a wide variety of functions. It is awesome. RNA is the new black

81. Unlike DNA, RNA is capable of forming a wide variety of shapes. This is due in part to the fact that RNA is generally single stranded: without having to base pair specifically along the entire length of the molecule, RNA is capable of forming hairpins, loops, and other structures when it selectively pairs with itself. Additionally, hydrogen bonds along the backbone of the molecule contribute to the overall diverse three dimensionality of RNAs.

82. Perhaps one of the best known RNA molecules, transfer RNA (tRNA) forms a cloverleaf set of loops. The final, 3D look of tRNAs looks somewhat akin to an electric drill. At one end of the drill, there is an anticodon that matches up to a three letter sequence in mRNA. The other end carries the corresponding amino acid according to the genetic code.

83. RNA is capable of forming complexes with protein. the ribosome, a multi-unit piece of equipment designed to synthesize proteins, is composed of 4 RNA molecules and over 80 different proteins.

84. Certain RNAs are capable of catalysis. Ribozymes can cut or ligate other RNA moelcules, catalyse peptide bond formation in the ribosome, etc.

85. siRNA siRNA binds RISC RNAs can be regulatory. In this example, silencing RNA (siRNA), when processed by the RISC complex, is capable of binding to a very specific target which ultimately leads to the target mRNA being degraded. This allows for highly specific knock down of protein production. siRNA unwinds ss siRNA binds target target mRNA is degraded Image from Santa Cruz Biotechnology

86. protein

87. Proteins are made from amino acids. These molecules contain an amine group, a carboxyilic acid group and a variable side chain (marked as R). The arrangement of these groups is centered around an alpha carbon, which is chiral (a fancy way of saying that the molecule does not have a super imposable mirror image) – this creates D- and L- amino acids. Traditionally, only L-amino acids are found in proteins.

88. Amino acids are attached via peptide bonds to create a polypeptide chain. Peptide bonds are covalent bonds that require a chemical reaction to break. polypeptide chain peptide bond

89. acidic basic non-polar polar Amino acids can be classified into four major groups. These groups provide a rough guideline for how these amino acids function. All in all, there are 20 standard amino acids.

90. polypeptide chain The combination of different amino acids into a polypeptide chain creates the primary sequence of a protein. Although any combination is theoretically possible, the chemical properties of the various R groups do limit the likely sequences. However, there are thousands of different proteins in the human body!

91. Polypeptide chains are capable of folding into specific structures thanks to a variety of non-covalent interactions. These include hydrogen bonds, electrostatic attraction and van der Waals interactions. Each of these is less strong than a covalent bond, but combined can stabilize a three dimensional structure.

92. The unfolded polypeptide (held together by covalent bonds) forms a three dimensional secondary structure using non-covalent interactions. As the protein folds, hydrophobic (water hating) and hydropohillic (water loving) regions of a protein are formed based on the type of side chain and overall conformation. The exact, atomic level 3D structure is known as the tertiary structure. Proteins oftentimes have a hydrophobic core – it is energetically more favorable to put all of the hydrophobic amino acids together in the interior of the protein, which is one reason why a 3D structure is a stable conformation.

93. The alpha helix is a classic secondary structure that forms a right-handed coil. It is characterized by its overall diameter, number of residues per turn and the height of each full turn.

94. The beta sheet is another common secondary structure, which is formed by extended strands of a polypeptide connected laterally by hydrogen bonds. Polypeptide chains do have a directionality (amino terminal to carboxyl terminal), and beta sheets can form parallel or anti-parallel orientations.

95. The beta sheet is another common secondary structure, which is formed by extended strands of a polypeptide connected laterally by hydrogen bonds. Polypeptide chains do have a directionality (amino terminal to carboxyl terminal), and beta sheets can form parallel or anti-parallel orientations.

96. Proteins can contain a combination of alpha helicies and beta sheets in the same polypeptide. The final 3D conformation represents the tertiary structure. Proteins are also capable of forming complexes with other proteins, creating quaternary structure.

97. Primary Structure: The amino acid sequence, essentially representing what is covalently bonded together 1 Secondary Structure: localized areas of folding, commonly alpha helicies or beta sheets. Formed with non-covalent bonds 2 3 Tertiary Structure: the complete, atomic-level structure of a 3D protein Quaternary Structure: arrangement of multiple folded proteins into a larger order complex 4 http://www.contexo.info/DNA_Basics/images/proteinstructuresweb.gif

98. Proteins come in all sorts of shapes and sizes, and are capable of performing many tasks. Some, such as collagen, are structural. Others, like calmodulin, are involved with complex signaling networks that regulate multiple functions. Enzymes are often considered the molecular machines , as they are capable of catalyzing specific reactions.

99. special proteins: antibodies

100. Antibodies, also known as immunoglobulins, are large proteins that identify foreign objects and play a major role in our immune system. The antibody is unique in that it has highly specific antigen-binding sites that recognize a wide variety of targets.

101. The main “y” shaped body of the an antibody is relatively similar to all antibodies; the antigen binding site is extremely diverse. A human creates billions of different antibodies, each capable of binding with high specificity to a foreign molecule.

102. special proteins: enzymes

103. Enzymes are proteins that catalyze chemical reactions. Substrates (starting material) bind to the enzyme which performs a specific function. Product is released. Enzymes are specific for substrates and greatly speed up reaction rates. The example above demonstrates how sucrose is broken into glucose and fructose during digestion.

104. Enzymes work in part because they very specifically bind to a target (called a ligand). This prevents the enzyme from performing spurious reactions.

105. This is an example of an enzyme binding site: the ligand is shown in pink. As you can see, the protein molecule is organized in such a way to specifically bind the substrate with hydrogen bonds and electrostatic interactions.

106. Lesson in a Tweet: Legos have nothing on DNA, RNA and Protein.

107. the care and feeding of your DNA

108. replication

109. replication DNA needs to be replicated with high fidelity and at great speed during the course of cell division. The entire human genome gets replicated in just a couple of hours – over 3 million base pairs!

110. DNA replication can occur quickly and accurately due to the process of hybridization. Remember, A can only base pair with T; G can only base pair with C. This means that with only one strand, you can easily build the complimentary strand.

111. DNA synthesis is semi-conservative. From the parent DNA strand, the daughter strands will each have on strand from the parent and a newly synthesized complimentary strand.

112. In order for DNA replication to occur, origins of replication must be unwound by specific proteins to reveal ssDNA

113. One of the proteins necessary for DNA replication is DNA helicase. Biochemists have a sense of humor. Not a great sense of humor, but at least we try… Image from Snorgtees.com

114. DNA synthesis occurs from a 5’ to a 3’ direction. DNA polymerase adds base pairs in this direction, but can go backwards to check for incorrectly added bases – a proofreading function. Once an incorrect base has been removed, it can add the correct nucleotide and continue replication in a 5’ to 3’ direction.

115. Replication can occur quickly, at roughly 1000 base pairs per second in prokaryotes and ~50 per second in eukaryotes. However, even at that pace it would take too long to replicate an entire genome with only one start site. To make the process faster, replication can occur at multiple places in the genome. As the strands are separated and synthesis occurs, bubbles of replication develop and propagate along the DNA.

116. Replication is complex and requires a large number of proteins working together to accomplish everything in a smooth, coordinated fashion.

117. Complete replication of the human genome can occur in just a few hours! Image from Berkeley Open Computing Facility

118. repair

119. Simply replicating DNA isn’t enough – our DNA is constantly being damaged. Image from R&D Systems

120. Even a single incorrect base can have a serious effect. Sickle cell anemia comes from a single A to T mutation

121. DNA replication by itself is very efficient, due in part to the proofreading activity of DNA polymerase. However, addition of DNA replication with repair mechanisms make our care and feeding of DNA a highly efficient system. Much better than the US Postal System.

122. mismatch repair

123. There are many different types of repair, one of which is mismatch repair. When a DNA mismatch has been identified, a series of proteins are involved with removal of a stretch of the newly synthesized DNA which is subsequently replaced. It is believed that mismatch repair systems recognize the new strand by the presence of nicks in the that have not yet been ligated together.

124. base/nucleotide excision repair

125. These are just two of the many ways in which our DNA can be damaged. Dupurination occurs spontaneously thousands of time per second in our bodies, and if left unrepaired would cause deletions in the genome. Other damage include those created by UV such as the creation of thymine dimers.

126. This is a schematic of nucleotide excision repair. In this example, the damaged region has been recognized by a specific enzyme and replaced using the bottom strand as a template. The process of damaged base pair recognition can be quite fascinating, oftentimes recognising distortions in the normal structure of the DNA double helix: in some cases, the enzyme gently squeezes the double stranded DNA, causing incorrect bases to flip out of the double helix!

127. double strand break repair

128. Double strand breaks are repaired using a variety of functions, including homologus recombination. This requires the presence of an almost identical sequence to be used as a template for the repair.

129. Lesson in a Tweet: It’s amazing we are as normal as we are. Well, at least some of us.

130. trancription

131. Now we’re going to take a look at transcription, the process by which a DNA sequence is transcribed into a coding RNA sequence.

132. DNA C A T G RNA C A U G Recall that RNA has a very similar structure to that of DNA, the primary difference being the sugar used in the backbone and the change of a Thymine in DNA to a Uracil in RNA.

133. start site stop site gene of interest template strand promoter terminator DNA is organized into genes, which have promoter and terminator regions to facilitate the start and stop of translation. Note, the sequence of the gene of interest has directionality; the template strand is the compliment to the coding sequence.

134. Transcription is complicated! Before it can start, several proteins must bind, including RNA Polymerase which actually synthesizes the new RNA strand, along with many mediating proteins (transcription factors) and activators.

135. But wait! Before RNA polymerase and other transcription factors can bind, the gene of interest must be released from its tight storage. Histone modifying enzymes and chromatin remodeling complexes allow for the regulatory sequences of the gene to be accessible.

136. Regulatory DNA sequences far upstream of the gene (sometimes thousands and thousands of base pairs upstream) need to be organized properly for transcription to start. Transcription requires complex spatial organization of DNA and proteins to begin.

137. As RNA Polymerase works, it uses the template strand of DNA to construct a complimentary RNA strand using RNA nucleotides (UTP, ATP, CTP, GTP). This is known as messenger RNA (mRNA) During this process, RNA Polymerase itself is phosphorylated (the chemical addition of phosphate to the protein), which allows it to leave the pre-initiation complex and move along the DNA strand.

138. The phosphorylated RNA polymerase is capable of binding proteins involved with the processing of the RNA.

139. In order for RNA to mature, it needs to have a 5’ cap and a poly-A tail added. These modifications help prevent degradation of the mRNA as well as proper export of mRNA from the nucleus to the cytoplasm, where translation occurs.

140. Remember that eukaryotic genes have introns that are excised from the mature RNA molecule which is made of exons that have been stitched together.

141. Introns are recognized by special sequences at the beginning and end of an intron that ultimately lead to the excision of that portion of the newly synthesized RNA molecule.

142. The splicing event involves a complex of molecules known as the spliceosome. Note: the spliceosome is composed of primarily RNA molecules rather than proteins. The spliceosome loops oiut the introns, creating a lariat of non-coding RNA.

143. In a process known as alternative splicing, a single gene can make several different mature mRNA products based on how the exons are organized.

144. After mRNA has been capped, polyadenylated and spliced, it is exported from the nucleus to the cytosol where translation can occur.

145. Many different mRNAs can be produced off of a single gene at any given time. The electron micrograph shown below depicts several newly synthesized mRNA strands being made off of two different genes.

146. translation

147. Translation is the process by which proteins are constructed by reading the mRNA.

148. The genetic code is used to translate nucleotides into specific amino acids. Triplets of nucleotides code for one amino acid. Note, the genetic code is redundant – oftentimes, many different codons code for the same amino acid.

149. The genetic code is read in groups of three nucleotides. It is extremely important that the correct reading frame is chosen. In the example to the left, the same sequence can be read in three different ways, depending on when you choose to start the first triplet. The results are all very different!

150. One way that cells have devised to ensure correct reading frames is to always start a protein with a Methionine, which therefore means that a mRNA reading frame must always start with AUG.

151. Like mRNA synthesis from DNA, many different amino acid chains can be read off of a single mRNA molecule. The electron micrograph below shows many protein production machines (ribosomes) arranged along one mRNA.

152. Transfer RNA (tRNA) reads the mRNA triplets and attaches the correct amino acid. tRNA are small RNA molecules that form a unique three dimensional structure. At one end of the tRNA, the anticodon base pairs with the codon in the target mRNA (shown in blue). The other end of the tRNA contains the appropriate amino acid (shown in green). The blue cartoon to the far right will represent tRNAs in subsequent figures.

153. Each tRNA has a specific enzyme that recognizes both the codon and the appropriate amino acid. Recall that enzymes are highly specific: this ensures that the correct amino acid is attached to the correct codon.

154. Ribosomes are the protein manufacturing machines of the cell. The ribosome is a large complex made from more than 50 proteins and 4 RNA molecules.

155. The Ribosome has three functional sites: the A-site binds tRNA, the P-site houses the tRNA attached to the growing peptide chain, and the E-site holds the empty tRNA before it is released.

156. How a ribosome works: Step 1 As the ribosome slides along the mRNA, the growing chain is located on the P site. The next codon to be read occupies the A site, where a newly bound tRNA that corresponds to the correct codon has been positioned.

157. How a ribosome works: Step 2 The ribosome enzymatically couples the newly growing peptide chain to the A-site, extending the protein by one amino acid.

158. How a ribosome works: Step 3 Next, the large subunit translocates one codon.

159. How a ribosome works: Step 4 Finally, the small subunit translocates. This repositions the newly extended polypeptide chain into the P-site, and the empty tRNA into the E-position.

160. How a ribosome works: Back to step 1 Once the empty tRNA in the E-position has left, the next tRNA that aligns in the A-site can begin the cycle again.

161. As the polypeptide chain is being made, it begins to fold. This process is quite complex and can happen spontaneously or may require the assistance of large folding complexes. Non-covalent factors (such as hydrophobic and hydrophillic core formation and metal ion binding) oftentimes energetically drive reactions. Proteins can be further modified by a series of post-translational modifications (PTMs) or be catalytically processed before becoming mature.

162. I think protein folding is amazing, and I could easily spend a four hour class discussing this phenomenon. However, I know that this would cause most people to fall asleep immediately. I will relay one interesting fact: if proteins had to go through all of the potential formations before finding the correct conformation, it would take longer than the time the universe has been existence! The combination of energetic effects and folding machines dramatically decrease this time and allow proteins to fold quite quickly.

163. Lesson in a Tweet: transcription and translation made me the molecule I am today…

164. Genetics 101:

165. A little history… Blending vs particulate hypotheses of inheritance Blending: genetic material contributed by the two parents mixes in a manner analogous to colors (red + blue = purple) Particulate: parents pass on discrete heritable units that retain their separate identities in offspring

166. Blending hypothesis: Genetic material contributed by the two parents mixes like colors

167. Blending hypothesis: Evidence against A uniform population of individuals would result Other phenomena of inheritance (eg. traits skipping a generation) are not explained

168. Particulate hypothesis: Parents pass on discrete heritable units that retain their separate identities in offspring

169. Gregor Mendel: Guessed that sperm and eggs carry distinct “units” of information about heritable traits Gregor Mendel Sweet Pea Campbell & Reece, 14-1

170. Mendel used controlled breeding to analyze patterns of inheritance White flowers Purple flowers All plants had purple flowers 705 purple-flowered plants 224 white-flowered plants Campbell & Reece, 14-3

171. Mendel used controlled breeding to analyze patterns of inheritance Alternative versions (alleles) of genes account for variations in inherited characters For each character, an organism inherits two alleles (one maternal and one paternal) If the two alleles at a locus differ, then: The dominant allele determines the organism’s appearance The recessive allele has no noticeable effect on the organism’s appearance White flowers Purple flowers All plants had purple flowers 705 purple-flowered plants 224 white-flowered plants Campbell & Reece, 14-3

172. Alternative versions of genes account for variations in inherited characters Allele for purple flowers Locus for flower-color gene Campbell & Reece, 14-4 Allele for white flowers Allele: alternative versions of a gene Locus (plural loci): A gene’s specific location along the length of a chromosome

173. For each character, an organism inherits two alleles (maternal and paternal) If the two alleles at a locus differ, then: The dominant allele determines the organism’s appearance The recessive allele has no noticeable effect on the organism’s appearance P Generation Appearance: Purple flowers PP White flowers pp Genetic makeup: p P F1 Generation Appearance: Genetic makeup: Purple flowers Pp Campbell & Reece, 14-5

174. The outcome of a cross can be predicted using a Punnett square P Generation Genotype Phenotype Purple flowers PP White flowers pp Appearance: Genetic makeup: PP (homozygous) Purple 1 p P Gametes F1 Generation Pp (heterozygous) 3 Purple Appearance: Genetic makeup: Purple flowers Pp 2 p Gametes: 1 1 P 2 2 Pp (heterozygous) Purple F1 sperm p P F2 Generation P PP Pp pp (homozygous) White F1 eggs 1 1 p Pp pp Ratio 1:2:1 Ratio 3:1 Campbell & Reece, 14-6 3 : 1

175. The outcome of a cross can be predicted using a Punnett square P Generation Genotype Phenotype Purple flowers PP White flowers pp Appearance: Genetic makeup: PP (homozygous) Purple 1 p P Gametes F1 Generation Pp (heterozygous) 3 Purple Appearance: Genetic makeup: Purple flowers Pp 2 p Gametes: 1 1 P 2 2 Pp (heterozygous) Purple F1 sperm p P F2 Generation P PP Pp pp (homozygous) White F1 eggs 1 1 p Pp pp Ratio 1:2:1 Ratio 3:1 Campbell & Reece, 14-6 3 : 1

176. Alleles can show different degrees of dominance and recessiveness P Generation Red CRCR White CWCW Codominance Incomplete dominance Gametes CR CW P Generation Red RR White WW Gametes R W Pink CRCW F1 Generation Roan RW F1 Generation 1 1 Gametes CR CW 2 2 1 1 Gametes R W 2 2 Sperm 1 1 CR CW 2 2 Sperm Eggs 1 1 R W F2 Generation 2 2 Eggs F2 Generation 1 CR 2 1 R 2 CRCR CRCW RR RW 1 1 W CW 2 2 RW WW CRCW CWCW Campbell & Reece, 14-10

177. Phenotypes can be determined by more than one gene and vary in gradations Polygenic inheritance: an additive effect of two or more genes on a single phenotypic character Quantitative characters: those that vary in the population along a continuum (eg. height and skin color) Many phenotypes are determined by genotype and environment AaBbCc AaBbCc aabbcc Aabbcc AaBbcc AaBbCc AABbCc AABBCc AABBCC 20/64 15/64 Fraction of progeny 6/64 1/64 Campbell & Reece, 14-13

178. Mendel used controlled breeding to analyze patterns of inheritance We can’t do that in humans! We can study existing families We can use model organisms

179. A pedigree is a family tree that describes the inter-relationships of parents and children across generations Key Affected male Male Mating Offspring, in birth order (first-born on left) Affected female Female 1st generation (grandparents) Ww Ww ww ww 2nd generation (parents, aunts, and uncles) Ww Ww Ww ww ww ww 3rd generation (two sisters) Inheritance patterns of particular traits can be traced and described using pedigrees Pedigrees and laws of probability can be used to make predictions about future offspring WW ww or Ww No widow’s peak Widow’s peak (a) Is a widow’s peak a dominant or recessive trait?

180. A pedigree is a family tree that describes the inter-relationships of parents and children across generations Key Affected male Male Mating Offspring, in birth order (first-born on left) Affected female Female 1st generation (grandparents) Ww Ww ww ww 2nd generation (parents, aunts, and uncles) Ww Ww Ww ww ww ww 3rd generation (two sisters) Inheritance patterns of particular traits can be traced and described using pedigrees Pedigrees and laws of probability can be used to make predictions about future offspring WW ww or Ww No widow’s peak Widow’s peak (a) Is a widow’s peak a dominant or recessive trait? 1st generation (grandparents) Ff Ff Ff ff 2nd generation (parents, aunts, and uncles) ff ff Ff Ff FF or Ff ff 3rd generation (two sisters) FF ff or Ff Attached earlobe Free earlobe (b) Is an attached earlobe a dominant or recessive trait?

181. Thousands of disorders are known to be inherited as simple recessive traits Sickle-cell disease affects one out of 400 African Americans, ~1/10 have sickle-cell trait In low oxygen conditions, the cells lose their healthy round shape and become sickle-shaped, causing them to get stuck in capillaries Typically the disease results in organ damage and premature death. Red blood cell shape Normal cells are full of individual hemoglobin molecules, each carrying oxygen 10 µm Fibers of abnormal hemoglobin deform cell into sickle shape Campbell & Reece, 5-22

183. Some human disorders are caused by dominant alleles Dominant alleles that cause a lethal disease are rare and often arise by de novo mutations OsteogenesisImperfecta (brittle bone disease), 35% of cases are caused by new mutations Dominant alleles can be propagated if defects exhibit late in life Huntington’s Disease (a degenerative disease of the nervous system), has no obvious phenotypic effects until the individual is about 35 to 40 years of age

184. A preventive approach to simple Mendelian disorders is possible Using family histories, genetic counselors help couples determine the odds that their children will have genetic disorders For a growing number of diseases, tests are available that identify carriers and help define the odds more accurately In some cases, targeted therapies are being developed

185. Genetics 101: Lesson in a Tweet: Inheritance of genes + environment = life as we know it

186. small molecules

187. We’ve briefly discussed how nucleotides make up DNA and how amino acids are strung together into proteins. Now we’ll cover some other molecules important to cellular function.

188. water Perhaps one of the most commonly overlooked molecules is water. Cellular life is aqueous (our cells are bags of water), and is made possible through the unique properties of H2O.

189. Water is, of course, made of two hydrogen molecules (shown in white) and one oxygen molecule (shown here in red). Note that the structure of water is not linear: there are unique angles between the bonded atoms.

190. Water is slightly polar, with regions that are slightly electronegative and regions that are slightly electropositive. This means that water molecules can align in solution according to its polarity (weak, non-covalent bonds are shown in the right hand cartoon as red lines). These alignments are weak and transient – in fact, water molecule alignment is thought to flicker constantly in solution.

191. Bonds have defined lengths. In a vacuum or in water, bonds have differing strengths. What is important in this table are not the absolute values of bonds, but their relative values: in water, the ionic, hydrogen and van der Waals interactions are much closer in strength. This means that several hydrogen bonds can closely approximate an ionic bond. In a vacuum, this is not the case. The relative change of bond strength from a vacuum to water allows cellular life to work on an energetic level.

192. hydrophilic Molecules that can be easily surrounded by water are hydrophilic (meaning water loving). Note how the water molecules use their polarity to surround the above molecules.

193. hydrophobic Molecules that are not polar do not dissolve in water. Note how the water forms a cage around the above molecule – this is like an oil droplet in water.

194. sugar My favorite molecules.

195. Image from Wikipedia courtesy of LauriAndler

196. Sugars are carbohydrates (hydrates of carbon). Cx(H2O)y, where x is at least 3.

197. Depending on the arrangement of the double bonded oxygen, sugars are classified as either aldoses or ketoses. The number of carbons is related to the sugar family (i.e. pentoses).

198. Sugars easily and quite frequently form ringed structures. Note that the position of the hydroxy groups (-OH) are important, as their three dimensional orientation are essential to their recognition and function. Two of my favorite sugars: glucose, which is the major storage molecule in the cell, and ribose which makes up the backbone for DNA and RNA.

199. Sugars can be combined together to create disaccharides.

200. Sugars can also be used to create large, complex structures that have a variety of uses.

201. The large sugar structures can be used for energy storage. Note, the protein in the middle is of small stature when compared to the glycogen surrounding it. Image from Wikipedia.com c/o MikaelHaggstrom

202. lipids Lipids are an essential component of membranes, as well as being essential to energy storage and signaling in cells.

203. Lipids are defined as molecules that have a hydrophobic component. In this case, the lipid has a long, hydrodrabon tail as well as a hydrophilic head.

204. Three lipids can be combined with a glycerol molecule to form a fatty acid. Lipids that are straight chains are called saturated, while lipids that have kinks (formed by double bonds) are called unsaturated fatty acids. The overall shape of the fatty acid is a factor in their function.

205. Since fatty acids have both a hydrophilic and hydrophobic component, when combiend with water they will either form a layer at the top of the solution, or a self-contained bubble of fatty acid called a micelle. These form spontaneously.

206. Phospholipids are specialized fatty acids that contain a phosphate in the hydrophilic region of the molecue. Phospholipids can form a bilayer, which is the major component of any membrane found in a cell. Membranes have many added molecules such as protein pores and chemicals.

207. Other lipids include cholesterol and some steroids. They are primarily hydrophobic molecules with small hydrophilic areas.

208. Fat soluble vitamins such as vitamins A, D, E and K are also lipids. Image from PerriconeMD.com

209. other molecules of interest… The cell is packed with all sorts of amazing molecules that perform a variety of unique functions.

210. Adenosine triphosphate(ATP) is the major energy carrying molecule of the cell. Image from sacredwaste.com

211. In ATP, energy derived from sunlight or food is stored in a high-energy phosphate bond created by adding a free phosphate to ADP. ATP stores the energy until it is released under specific catalytic conditions.

212. Other molecules of interest include ubiquitin, which is a post-translational molecule that often tags proteins for death; Combinations of post-translational modification such as ubiquinylation, phosphorylation and methylation are used to create complex signals in cells. Images from zazzle.com, The Shechter Lab, and cafepress.com

213. Heavy Metals are also important.

214. OK, actually there are very few heavy metals in the average cell. 99% of all cellular elements are C, N, O and H. The blue elements comprise an additional 0.9%, while the green and yellow molecules are only found in trace amounts. However, the green molecules are known to be absolutely necessary for cellular function.

215. The average cell is mostly water, with proteins comprising the majority of the macromolecule population.

216. Lesson in a Tweet: H2O is the formula for water, what is the formula for ice? H2O cubed #chemistryisfunny

217. From cell… To organism

218. Reviewing the cell

219. The gene products a cell makes define its identity and function Muscle cells have many fibers that allow them to contract Pancreatic b cells make insulin to regulate blood sugar Red blood cells make hemoglobin to carry oxygen

220. Multiple cells types work together to form a tissue – CIP, the pancreas The pancreas secretes three main types of molecules: Hormones (inc. insulin) to regulate blood sugar Bicarbonate (baking soda) to neutralize stomach acid Enzymes to help digest food (Somatostatin) (Digestive enzymes) (Glucagon) (Insulin)

221. Multiple tissues work together to form an organism – CIP, digestion Every organism needs nutrients from the environment to survive, and digestion is complicated! More than 10 tissues coordinate to extract and absorb nutrients from food, and get rid of the rest

222. From cell… To organism Lesson in a Tweet: Organisms are made of tissues made of cells made of small molecules

223. Translational Medicine What happens when the organism does not function properly?

224. Biochemical basis of disease Why study this? Knowing when and where a gene is expressed and the general function of the gene product can help us: Understand normal function Understand pathogenesis Devise therapies

225. Targeted Therapies Gleevec is our poster child Gleevec treats chronic myelogenous leukemia Effective with minimal side effects In order to make this drug, decades of research came before

226. Targeted Therapies Gleevec is our poster child Gleevec treats chronic myelogenous leukemia Effective with minimal side effects In order to make this drug, decades of research came before Define the disease in humans Identify the molecular cause Use knowledge gained from basic science to find an appropriate target Design and test potential drugs Take it to the patients

227. Image from snorgtees.com

228. The table above represents stats from the OMIM database as of August 2011. There are many genes and phenotypes that have been correlated, although there are many that still are unknown. In essence, we’ve only begun to understand the complexity of genotype/phenotype relationships.

229. Image from remedymd.com Translational science is meant to accelerate the collaboration between a variety of organizations to facilitate scientific discovery to health care application timelines. As all of the above areas overlap, it becomes clear that collaboration is essential.

230. The NIH began the Clinical & Translational Science Awards several years ago to create centers designed to facilitate translational research. The CTSA awards will be administered by the new National Center for Advancing Translational Sciences (NCAT). The following quotes are from a paper by the NIH director abound NCAT and translational science.

231. NCATS’s mission is to catalyze the generation of innovative methods and technologies that will enhance the development, testing, and implementation of diagnostics, therapeutics, and devices across a wide range of human diseases and conditions. Collins, FS. Science Translational Medicine 6 July 2011

232. Therapeutics of the future likely will be designed with cellular networks in mind, rather than being limited by historical designations of disease category. Collins, FS. Science Translational Medicine 6 July 2011

233. …little focused effort has been devoted to the translational process itself as a scientific problem amenable to innovation. Collins, FS. Science Translational Medicine 6 July 2011

234. Lesson in a Tweet (or two): A mouse is not a human, but they’re pretty close you say translational, I say translational, let’s all get funding.

235. Techniques

236. Southern Hybridization Blood, tissue, or cultured cells Agarose gel of separated DNA Genomic DNA Extract DNA Separate via electrophoresis Transfer to membrane Hybridize with radioactive probe and expose to film Transfer apparatus Southern Blot

238. Probe with antibodiesHybridize with radioactive probe and expose to film Transfer apparatus Southern Blot

239. imaging

240. Light microscopy can be used to observe phenomenom on a micrometer scale. Depending on how you shine the light and what filters you use will change the quality of the picture.

241. Fluorescent microsopy uses special compounds call fulophores that emit light when a specific wavelength is directed at them. Molecues can either have fluorphores attached to them or antibodies with attached fluorphores can be added to localize specific cellular entities. Advances in mathematics can “deconvolute” images to create very high resolution images (see panel A and B in green above)

242. To visualize samples at an atomic level, electron microscopy shoots a beam of electrons at a sample.

243. Scanning electron microscopy creates high resolution images of the surface of molecules. Shown here is a hair cell.

244. NMR is a technique that uses magnetic resonance to create models of atomic structure.

245. X-ray crystallography is another technique to generate atomic resolution structures. This is one of my favorite techniques!

246. finding genetic abnormalities: FISH PCR

247. Hybridization, the highly specific and efficient process by which molecules can recognize and re-align with each other, is an important part of many molecular biology techniques

248. In this schematic, the probe DNA (in red) was labeled with fluorescent probes (the white circles). Target DNA (shown in blue) AND the probe DNA are deanatured using high temperature, and allowed to re-anneal. At this point, the probe is capable of specifically binding to target stretches of DNA. Image from Nature Reviews Genetics

249. The fluorescent probes can be many different colors, and can be used to “paint” specific DNA areas. The above image is an example of chromosome painting. Image modified from Nature Reviews Genetics

250. bcr/abl rearrangment In the case of bcr/abl, a translocation puts the green section of a gene immediately adjacent to a red section that would normally be on a different chromosome. This results in a hybrid protein not normally found in the human body, and ultimately results in a severe disease. In the above example, you can see how the normal painting of the chromosomes is disrupted by large translocations of DNA (resulting in chromosomes that are clearly part yellow and part red). Image modified from RERF.org and Wikipedia

251. Polymerase Chain Reaction (PCR) is a technique which uses specific DNA primers to amplify a section of DNA. This process is specific, iterative, and can take a single strand of DNA and create millions of replicates in a short period of time. In this way, a small amount of DNA can be “amplified” to generate enough sample for sequencing or other forms of analysis.

252. Image from www.csi.us.ubi.com PCR shows up a lot in CSI. A lot.

253. Crime shows generally use PCR as part of a process known as DNA fingerprinting. In everybody’s genome, there are regions of DNA that are comprised of short tandem repeats. The sequence of many of these repeats is known, so we can create primers to match them. What differs between humans are the number of repeats. Once a chosen STR locus has been amplified, it can be separated by size (one such method is called electrophoresis). A human generally has different sized STR loci : the paternal and maternal chromosomes are generally different.

254. Using several different STR loci, you can develop a profile of a person based entierely on the sizes of their STR loci. You can compare these loci between individuals

255. CODIS uses 3 different STR Loci to create a DNA fingerprint. With 13 loci, the chances of an identical DNA fingerprint is about 1 in one billion.

256. DNA sequencing used to be an arduous task ; however, modern technology has greatly increased the speed while decreasing the cost of sequencing. The image shown here is a readout of a sequencing reaction. Image from University of Chicago Cancer Research Center DNA Sequencing Facility

257. Genetic mutations can be found through sequencing. Shown here, the upper panels contain wild type sequence; the lower panel shows a sequence that has mutations. Image from University of Chicago Cancer Research Center DNA Sequencing Facility

258. Lesson in a Tweet: you can’t hide from PCR. Technology is #winning!

259. so what? Now we are going to choose an interesting genotyp/phenotype correlation as a topic and walk through several common biological databases.

260. Early onset Alzheimer's disease (AD) is a genetic disorder.

261. Apolipoprotein E APOE It has been linked to the apolipoprotein E (APOE).

262. Cys130Arg APOE Specifically, a mutation that causes a Cys at position 130 to mutate into an Arg…

263. CU APOE This has been shown to arise from a mutation of a single C to a U in mRNA…

264. CT APOE … which came from a somatic mutation of a C to a T in the APOE gene.

265. APOE So what’s APOE?

266. APOE is one of several lipoproteins that are found in chylomicrons,which serve as vessels for transporting lipids in the body. Image from The Ladu Lab

267. Most of us are familiar with hearing about good and bad lipids: these are derived from the composition of various types of chylomicrons that circulate in our bodies. Image from Medscape.com

268. Lipid metabolism begins in the small intestine, where immature chylomicrons pick up partially processed lipids. Eventually, lipids get processed in the liver and delivered through the body via the blood stream. small intestine LIVER lymphatic system APOE is generally only found in chylocmirons that circulate in the blood stream. blood stream Image modified from the Journal of Undergraduate Biological Studies

269. APOE However, that isn’t the whole APOE story – APOE has been found to be important in many different cellular processes including many involved with proper neuronal development, function and signaling. However, the exact details about how APOE functions in these capacities is unknown. Image from nature Reviews Neuroscience

270. There are four major variants of the APOE gene. They vary in the AA produced at position 112 and 158. APOE is found on the long arm of chromosome 19. Image from nature Reviews Neuroscience;

271. For the observant: you may have noticed that originally I was talking about Cys130Arg, but the previous slide discusses Cys 112Arg. Do I have problems counting? Yes and no. The Cys is residue 130 if you include every single amino acid that the mRNA codes for. However, during the processing of the protein, the leader sequence gets cut off (this is particularly common for proteins that are excreted out of the cell). This shortens the protein by 18 residues. If you re-number the residues, this makes position 130 change to position 112.

272. Here’s the sequence of the gene… Image from UniProt

273. This is the amino acid sequence along with a schematic that depicts all of the known post-translational modifications (with table below) Images from UniProt

274. And this is the crystal structure of the APOE protein, with the Cys/.Arg mutation pointed out as a circle. Have I mentioned how much I like crystallography? Image from Huang, Y et al. 2001 PNAS 98(15):8838

275. so how did I get that information? so how do I get more information?

276. Gene and RefSeq Genome Maps Allelic Var/Disease Expression Homologus G/P Structure Answer: Databases, darling! I’m going to walk through several database that I commonly use when presented with a molecular question.

277. I like starting at the NCBI home page.

278. Let’s head on over to OMIM…

279. Let’s check out the entry on Alzheimer Disease 2.

280. The OMIM record includes a lot of information (see the extensive table of contents to the right). As this is a clinical record, there are sections for clinical features and synopsis. Although this does include information about genetics, more detailed info can be found in the genetic OMIM record.

281. OMIM is a curated, well referenced resource.

282. Now let’s check out the genetic reference in OMIM

283. Again, there are a lot of references. Of particular note, molecular genetics and allelic variants.

284. And once again, lots and lots of references.

285. Speaking of references, let’s check out PubMed.

286. PubMed has an auto suggest that will prompt you to check out the gene record for APOE . You can select homo sapiens directly from this window.

287. And like that, you’re in the Gene database. This database is a great hub for linking to a variety of other databases. We will often return to gene as a way to get to navigate to other databases.

288. Gene records are long. Really long.

289. The summary portion of any gene record provides a nice synopsis of the important information.

290. Let’s check out the table of contents. Specifically, let’s jump to the reference sequence heading.

291. Here the reference sequences at the genomic, mRNA and Protein level can be found.

292. You can also find the alternate genomic assemblies: The GRCh37 is the “original” human genome sequence issued by the Genome Reference Consortium. The Celera assembly was made up of 5 individuals and was issued by the company Celera. The HuRef genome is Craig Vetner’s personal genome.

293. Let’s take a look at the single nucleotide polymorphisms associated with this gene. (many databases here)

294. The SNP database depicts the genomic, transcript and protein mutations along with their relative frequency. It also links to all PubMed citations that reference a given SNP. Note: the Cys130Arg mutation is well characterized.

295. So let’s take a closer look at these genomic sequences… Back in gene, navigate to the genomic regions, transcripts and products heading.

296. Here you can choose the genome you wish to view (GRCh37, Celera or HuRef; identifiable by their acession numbers), and view the sequence using map viewer by clicking on “open full view”

297. The upper portion shows a zoomed out are of the genome. The little green chunks represent genes. The lavendar box shows the zoomed in region.

298. Below the larger map, the zoomed in area shows individual genes and well characterized variants.

299. The lowest level shows characterized SNPs, and links out to other databases such as the GWAS catalog.

300. Back in the gene record, we can also view the APOE gene using MapViewer.

301. MapViewer allows you to bring up multiple types of maps and compare them side to side.

302. ab initio modeling Ensemble Genes UniGene RefSeq The first map is derived from computer models. The remaining maps have been constructed and deposited into their representative databases: UniGene, Ensemble, RefSeq and Genes.

303. You can change the maps by clicking on the Maps & Options button. You can change the map to display similar genomic regions in other species.

304. This map compares chimp, mouse and human genomes. Homologous genes are connected by a line (not visible at this resolution).

305. Ever gene contains links to many different databases. This is another way to navigate around the NCBI databases. I’m going to head back over to OMIM.

306. Navigate over to Allelic Variants…

307. .0016 Here we can check out the SNP record that is associated with the Cys130Arg mutation.

308. Here you can find that the GRC and Reference assembly contain the wild type base, whereas the Celera and HuRef genomes contain the mutant allele. I can click on the chromosome position link from HuRef to look at this sequence.

309. We are now in Sequence Viewer, looking at the HuRef genome. I can click on sequence to absolutely locate the point mutation.

310. This shows a zoomed in sequence, along with the amino acid translation. Here you can see the Arg mutation. This is not good news for Craig Vetner.

311. Does having the CysArg mutation always result in early onset Alzheimer's? Let’s see if other organisms carry this mutation. Head back to Gene and then link to the UniGene database. (many databases here)

312. UniGene is a record of transcription, and provides two methods to seeing where a gene is expressed.

313. The first of which is EST Profiles. Essentially, this counts expressed sequence tags and can be used as a rough measurement of gene expression.

314. Disease State BODY SITES These results are broken down into three major categories: expression by body sites, expression during diseases states and expression during development. Development

315. GEO profiles is a collection of gene expression arrays, and represents a much more quantitative level of analysis.

316. In GEO, you need to find a record that includes analysis of your gene of interest in a relevant state (i.e. normal human being vs disease state)

317. liver brain Here we are observing expression levels in specific areas. Note, the highest levels are in the liver, but there is broad expression in the brain. This makes sense as APOE has been implicated in AD.

318. Let’s check out homologues in the HomoloGene catalog. (many databases here)

319. Here there are pre-computed BLAST searches between the major organisms (in this case, humans and chimps)

320. Here you can see a protein alignment, where you may note that the regular chimp sequence carries an Arg at the relevant position!

321. Let’s see if this holds true for other organisms. The Giant Panda was recently sequenced,,, let’s check it out. Head back to the Gene record and navigate to reference sequences.

322. Click on the reference protein sequence. We will use this to BLAST the panda genome.

323. In the protein record, there is a link to run a BLAST sequence off of the record in the right hand menu bar.

324. I’ve changed the parameters to search the giant panda database…

325. The BLAST record shows alignments in cartoon form. Even more alignments…

326. Low and behold, there is a predicted panda APOE. Click on the link to jump to the amino acid alignment.

327. Pandas carry the Arginine… This is actually true for all of the organisms I checked. But Pandas and chimps don’t get AD (that we know of)… so what does this all mean? Chances are there isn’t a structural defect created by the Arg mutant, otherwise it wouldn’t be tolerated in other organisms. To check this, I’m going to take a look at the protein structure.

328. Back at the Protein record, navigate to the Protein Structures summary section.

329. Here you will find structures that share sequence similarity to APOE. If your protein of interest has a structure, that’s great! If not, the related structures search will find structures from related sequences or domains.

330. Hovering over a section will bring up a window of more information. Click on the record to get more information.

331. This structure carries the Arg at position 112. Let’s view it in Cn3D

332. This is the default “wire frame” view. I think it is fairly hideous, so I’m going to change settings.

333. Much better. The mutated residue is shown in yellow with a white arrow pointing towards it. Note, it doesn’t appear to be causing any major structural problems (local areas of unfolding), but let’s see if we can find a wilde type structure that carries the Cys at position 112. Click on the pink record label.

334. We are now in a the Structure database, looking at the summary. To find related structures (that will hopefully have a wild type Cys at 112), click on the pink bar. This will bring up related structures.

335. Here are several related structures. It happens that the fourth structure is hits all of our qualifications (human APOE fragment with a Cys at position 112). Let’s view an alignment between it and the structure with an Arg at position 112.

336. This shows an overlapping structure. The Arg or Cys is shown in yellow in the lower right hand corner. Note that there doesn’t appear to be a major structural difference between the two! This indicates that the Arg mutation does not structurally destabilize the protein, and it is unlikely that there is a structure/function problem related to this mutation. This makes sense, as animals carry the Arg with no serious problems.

337. so what? And that, ladies and gentlemen, is an exploration of how I investigate a molecular problem using NCBI databases. However, there are many, MANY different and equally excellent databases out there.

338. Here are a few. They all have different areas of emphasis.

339. Of interest, BioGPS allows you to set up a personalized map of molecular databases. I think this has a lot of potential, and I will investigate it further – it may be a future CE class!

340. Apolipoprotein E APOE So there you have it in a nutshell. Investigating a molecular question using modern databases. Science is awesome.

341. Lesson in a Tweet: You can never have too many databases.

342. REVIEW

344. Cells & the Central Dogma

345. Molecular Architecture of the Cell

346. The Care & Feeding of DNA

348. Small molecules

349. Big molecules (A&P)

350. Translational Medicine

353. Image © Jorge Cham, PhD Comics www.phdcomics.com

354. Results for Cell Biology Cell Biology Cells (P/E) Major Organelles Replication, Repair Transcription Translation ProkaryoteEukaryote Both cell types are incredibly diverse; eukaryotes have organelles. CellsAreStuffedWithStuff Organelles are membrane bound and perform specific functions. Major organelles include the nucleus, ER, Golgi, mitochondria and the cytoskeleton DNACopyCopyFixCopy DNA is faithfully and accurately copied at great speed; it is also constantly being repaired.

355. Results for Cell Biology Cell Biology Cells (P/E) Major Organelles Replication, Repair Transcription Translation DNAtoRNA DNA must be unpacked for transcription to occur; dsDNA to ssRNA; exported from nucleus. RNAtoProtein translation takes ssRNA to a three dimensional protein; uses the genetic code to translate codons to amino acids.

356. Results for Major Molecules Major Molecules doublehelix DNA = A, T, G & C; double helix design and info arranged into genes. Packed tightly until ready for use! DNA RNA Protein Other RNAisthenewBlack RNA = A, U G & C; forms flexible structures, must be formatted and edited, encodes info AND performs functions. Awesome. ProteinKicksAss Machines of the cell, proteins come in all shapes and sizes and do splendid things.

357. Results for Major Molecules Major Molecules DNA RNA Protein Other SuperSweet sugars can be complex molecules that are in DNA/RNA, used in signaling and taste great. LovinLipids Lipids are hydrophobic, can form membranes, and are generally underappreciated. Includes “fats”, steroids, some vitamins MoleculesAreDiverse There are many astounding molecules in the body, some of which are essential for life (or death).

358. Results for Genetics 101 MendelSays Eat your peas. Genetics 101 Genes and their inheritance HauteCoutureGenes Information, handed down, generation after generation without too much damage.

359. Results for The Big Picture NCBI We. Will. Blow. Your. Mind. The Big Picture Human Disease & Databases TranslationConnectsDots genes, genomes, proteins demonstrably lead to phenotypes: let’s make it lead to healthy phenotypes.

360. Science 101 Jackie Wirz, PhD Sylvia Nelsen, PhD

361. JW: All figures from my portion of the presentation were derived from Essential Cell Biology, 3rd edition, unless denoted otherwise. SN: All figures from my portion of the presentation were derived from Google Images.

362. thank you