Chapter 2 Genetics Notes PDF

2 – GENETICS 2.1 Genetics and Heredity Gene – DNA sequence coding for a single gene product (protein, rRNA or tRNA) Genome – the sum of all DNA in a cell Locus – the chromosomal location of a gene Homologous Chromosomes – chromosomes that code for the same s...

2 – GENETICS 2.1 Genetics and Heredity Gene – DNA sequence coding for a single gene product (protein, rRNA or tRNA) Genome – the sum of all DNA in a cell Locus – the chromosomal location of a gene Homologous Chromosomes – chromosomes that code for the same set of genes in diploid cells They may have different alleles, one received from each parent. Allele – one variant of a gene Genotype – the set of alleles possessed by an individual Phenotype – the observable characteristic(s) resulting from a genotype Homozygous – having 2 of the same allele for a gene Heterozygous – having 2 different alleles for a gene Complete Dominance – when a heterozygote has the phenotype of only 1 of the alleles Dominant – form of a trait expressed in a heterozygote HUMAN BLOOD TYPES Recessive - form of a trait not expressed in a heterozygote Genotype Phenotype AA Type A Incomplete Dominance – phenotypes of the progeny are blends of the parental phenotypes AO Type A (ex. snap dragons – homozygous red crossed with homozygous white gives pink progeny) BB Type B BO Type B Codominance – both alleles are completely expressed (ex. blood types – ABO) AB Type AB OO Type O Law of Segregation – the two alleles for a trait segregate into haploid gametes Law of Independent Assortment – genes assort independently to the progeny Linked Genes – Genes that lie on the same chromosome; they do not sort independently. True Breeding – Homozygous for a trait (offspring from self-fertilization remain uniform) Monohybrid Cross – a cross following only 2 variations of a single trait Dihybrid Cross – a cross following 2 variations of each of two traits Test Cross – the cross of an organism with the dominant phenotype with a homozygous recessive organism for the same trait. It allows for the determination of the first organism as being homozygous or heterozygous for the dominant trait. Pleiotropism – when an allele has multiple associated phenotypic effects Polygenism – when multiple genes affect a phenotype Penetrance – probability a particular genotype results in a particular phenotype Complete vs Incomplete Penetrance Expressivity – term describing the variation of phenotype for a specific genotype Epistasis – occurs when the expression of a gene is dependent upon another gene ChadsPrep.com 26 In peas, yellow (Y) is dominant to green (y) and round (R) is dominant to wrinkled (r). 1. Homozygous yellow peas are crossed with homozygous green peas. The F1 generation is then self-crossed. What will be the phenotypic ratios in the F2 generation? 2. How would you determine if a yellow pea plant was homozygous or heterozygous for color? 3. A yellow round pea plant that is heterozygous for both traits (YyRr) is self crossed. What are the phenotypic ratios in the progeny? YR Yr yR yr YR Yr yR yr ChadsPrep.com 27 Sex-linked Genes Humans have 22 pairs of autosomes and 1 pair of sex chromosomes. Sex Chromosomes – Pair of chromosomes responsible for sexual determination Female = XX Male = XY Autosomes – Non-sex chromosomes Y-Linked Traits – rare as there are very few genes on the Y-chromosome All Y-linked disorders are passed on to all male offspring (but to no female offspring) X-Linked Traits – males only receive a single copy of the X-chromosome from their mother X-inactivation – In every cell in females, one X-chromosome is inactivated being converted into a Barr body. This prevents overexpression of the genes on the X- chromosome. Which X-chromosome is inactivated is not uniform across different cells making females a mosaic of two cell populations. Turner Syndrome (X) – females have only a single X chromosome resulting from nondisjunction Kleinfelter Syndrome (XXY) – males have an extra X chromosome resulting from nondisjunction Mitochondrial Inheritance – Mitochondrial DNA doesn’t segregate during meiosis All mitochondrial DNA is inherited from the mother. Genetic disorders coded by mitochondrial DNA will be passed on to all offspring. 4. Color-blindness is the result of an X-linked recessive allele. What is the probability that a colorblind father and a normal mother (homozygous) have a colorblind child? 5. What is the probability that a heterozygous mother (a carrier) and a normal father have a colorblind daughter? A color blind son? ChadsPrep.com 28 Pedigrees Autosomal Dominant Autosomal Recessive X-Linked Dominant X-Linked Recessive Mitochondrial Inheritance ChadsPrep.com 29 2.2 DNA Structure & Replication CHROMOSOME ORGANIZATION PROKARYOTES EUKARYOTES One circular chromosome Many linear chromatin (supercoiled by DNA gyrase) (called chromosomes when condensed in mitosis) Wrapped around nucleosomes (histone octamers) Have centromeres and telomeres CHROMATIN EUCHROMATIN HETEROCHROMATIN Gene-rich Gene-poor Lightly packed Densely packed Often active genes (transcription) Largely inactive genes Chromatin Remodeling Histone Acetylation – increases transcription by decreasing DNA-histone attraction Histone Deacetylation – decreases transcription by increasing DNA-histone attraction Histone Methylation – can increase or decrease transcription DNA Structure DNA/RNA Bases Right-handed double helix (B-form) Strands are antiparallel and complimentary Strands are held together by H-bonding and base-stacking Nucleotides connected by phosphodiester bond ChadsPrep.com 30  DNA REPLICATION Replication begins at the Origin of Replication which is AT-rich Bacterial chromosomes have a single Origin of Replication INITIATION Eukaryotic chromosomes have multiple Origins of Replication A complex of proteins (helicase/primase/polymerase) binds at the origin. Helicase unzips DNA forming a replication fork. Topoisomerase relieves tension/reduces supercoiling ahead of the fork. Single-strand binding proteins bind/stabilize single stranded DNA. The single stranded regions serve as templates for DNA synthesis. Complimentary nucleotides will be incorporated into the new DNA strand. Primase adds RNA primers to the leading (once) and lagging strands (many). ELONGATION DNA Polymerase III adds DNA nucleotides to the RNA primer (3’ end). Elongation always occurs in the 5’→3’ direction. Elongation is continuous on the leading strand. Elongation is discontinuous on the lagging strand. New DNA fragments on the lagging strand are called Okazaki fragments. A second DNA polymerase replaces RNA primers with DNA. DNA ligase ‘seals the nick’ between DNA fragments. DNA replication terminates when replication forks converge. TERMINATION The replication machinery dissembles and dissociates from the DNA. DNA replication is bi-directional. It proceeds in both directions (two forks) from the origin. DNA replication is semiconservative. New DNA has one ‘parent’ and one ‘daughter’ strand. In eukaryotes, the ends of chromosomes are called telomeres. Telomeres are comprised of G- rich repetitive nucleotide sequences which are added by telomerase. They protect the ends of the chromosomes from being degraded by exonucleases. ChadsPrep.com 31 Mutations & DNA Repair A mutation occurs when the wrong nucleotide(s) is/are incorporated into a new DNA strand. Most mutations are deleterious to the cell. A low level of mutations occur naturally during replication. DNA polymerases have ‘proofreading’ ability to minimize the number of mutations. Mutagens are agents that cause mutations (chemicals, radiation, etc.). Carcinogens are agents that cause cancer. DNA REPAIR -Most DNA polymerases can proofread each nucleotide against the Proofreading template -Errors are removed and replaced Repair for mismatched nucleotides that have evaded proofreading 1. Mismatch and surrounding nucleotides are removed Mismatch (backbone is cut in 2 places). Repair 2. DNA Pol III replaces the removed nucleotides. 3. DNA ligase ‘seals the nick.’ DNA damage is repaired (such as thymine-thymine dimers). Nucleotide 1. Damaged DNA is removed (backbone is cut in 2 places). Excision 2. DNA Pol I replaces the removed nucleotides. Repair 3. DNA ligase ‘seals the nick.’ MUTATIONS Point Mutation Change of a single nucleotide Substitution Substitution of one nucleotide for another Transition Purine for purine; pyrimidine for pyrimidine Transversion Purine for pyrimidine; pyrimidine for purine Insertion The addition of one additional nucleotide Deletion The removal of one nucleotide Missense Mutation Point mutation leading to a codon for a different amino acid Nonsense Mutation Point mutation leading to a stop codon Silent Mutation Mutation that doesn’t result in a change in amino acid Frameshift Mutation Insertion/deletion leading to a change in reading frame of a gene Rare mutation in which a 3 nucleotide sequence is Triplet Repeat Expansion duplicated/repeated in the genome Chromosomal Mutations Deletions Loss of a portion of a chromosome Duplications Duplication of a region of a chromosome Inversions Occurs when a region of a chromosome is removed, inverted, and reinserted into a chromosome Translocations Occurs when a portion of a chromosome is broken off and joined to a different chromosome ChadsPrep.com 32 2.3 Transcription & Translation Central Dogma The genetic code is degenerate (multiple codons may code for the same amino acid). AUG = Start Codon UGA/UAA/UAG = Stop Codons (U Go Away / U Are Away / U Are Gone) REPLICATION TRANSCRIPTION TRANSLATION Start Site Origin of Replication Start Site Start Codon (AUG) Elongation DNA Polymerase RNA Polymerase Ribosome Enzyme Cytoplasm (prokaryotes) Cytoplasm (prokaryotes) Location Cytoplasm Nucleus (eukaryotes) Nucleus (eukaryotes) *Transcription and Translation both occur in the cytoplasm in prokaryotes and are coupled. ChadsPrep.com 33 Transcription Gene expression in eukaryotes is monocistronic (one gene per mRNA transcript). Gene expression in prokaryotes is often polycistronic (many genes in a single mRNA transcript). These genes are located in an operon, a cluster of genes with related function that are governed by a single promoter. TRANSCRIPTION RNA Polymerase binds to the promoter region and begins unzipping the DNA. This forms a ‘transcription bubble.’ INITIATION Promoter includes Pribnow Box (prokaryotes) or TATA box (eukaryotes) In eukaryotes, transcription factors are typically involved with recruiting RNA polymerase to the promoter. RNA Polymerase begins transcription at the start site (5’ → 3’ direction). Nucleotide triphosphates complimentary to the template strand are joined together via phosphodiester bonds. The breaking of a high energy phosphate ELONGATION bond releasing pyrophosphate provides the energy for transcription. Template = Noncoding = Antisense Nontemplate = Coding = Sense Transcription is terminated at a special termination sequence (terminator). TERMINATION mRNA is released and the DNA double helix reforms. ChadsPrep.com 34  POST-TRANSCRIPTIONAL MODIFICATION An altered nucleotide is attached at the 5’ of the mRNA transcript. 5’ CAP It protects from degradation and is involved in translation initiation. Up to 200 adenine residues are added to the 3’ end of the mRNA transcript 3’ Poly-A Tail by poly-A polymerase. This protects the mRNA from degradation and is necessary for nuclear export & translation. Introns are removed and exons are spliced together to form mature mRNA. Usually carried out by spliceosomes but self-splicing also occurs (ribozymes). Splicing Spliceosomes are comprised of snRNA/snRNPs. Alternative splicing can produce multiple proteins from a single gene. Translation Ribosomes, an rRNA/protein complex, are responsible for translating mRNA into proteins. In prokaryotes, ribosomes (70S) are composed of a small (30S) and a large (50S) subunit. In eukaryotes, ribosomes (80S) are composed of a small (40S) and a large (60S) subunit. In eukaryotes, ribosomal subunits are synthesized in the nucleolus but assembled in the cytosol. tRNA are responsible for bringing the correct amino acids to the ribosome. The anticodon of the tRNA is complimentary to a specific codon of the mRNA being translated. A tRNA with an amino acid attached is called a ‘charged’ tRNA or aminoacyl-tRNA. Aminoacyl-tRNA synthetases ‘charge’ tRNA with the appropriate amino acid (requires ATP). ChadsPrep.com 35 Ribosomes translocate across mRNA binding the appropriate tRNA that have anticodons that are complimentary to the codons of the mRNA. The ribosome catalyzes peptide bond formation between the amino acids brought to the ribosome by the bound tRNA. The ribosome has 3 tRNA binding sites: 1. P site (peptidyl) – binds tRNA with growing peptide chain 2. A site (aminoacyl) – binds tRNA bringing the next amino acid to be added 3. E site (exit) – binds tRNA that brought in the previous amino acid TRANSLATION An initiation complex forms comprised of the ribosome, mRNA, an initiator tRNA (tRNAmet in eukaryotes, tRNAfmet in prokaryotes), and initiation factors. INITIATION The small subunit binds the 5’ end of mRNA (5’ Cap in eukaryotes). The initiator tRNA is bound in the P site. Initiation is completed by binding of the large subunit. New charged tRNA is bound at the A site (requires GTP). ELONGATION Peptidyl transferase (in the large subunit) catalyzes peptide bond formation. The ribosome translocates along the mRNA A→P→E (requires GTP) Release factors bind when a stop codon appears in the A site releasing the TERMINATION newly made polypeptide. Chaperone proteins may be involved in helping the newly synthesized protein properly fold. Proteins that start with a signal sequence are bound by the signal recognition particle (SRP) and docked to the ER. The growing peptide is passed into the lumen of the ER as it is produced. Post-translational modifications may be made at the ER or Golgi body. ChadsPrep.com 36  GENE EXPRESSION IN PROKARYOTES VS EUKARYTOES PROKARYOTES EUKARYOTES Polycistronic Monocistronic GENES/mRNA (Many genes/mRNA) (1 gene/mRNA) No splicing, no introns Introns are spliced out SPLICING (exceptions in archae) None Splicing POSTTRANSCRIPTIONAL 5’ Cap MODIFICATION Poly-A Tail Transcription/translation are Transcription in the nucleus TRANSCRIPTION/ coupled in the cytoplasm Translation in the cytoplasm TRANSLATION (Translation begins before transcription is complete.) MUTATIONS Point Mutation Change of a single nucleotide Substitution Substitution of one nucleotide for another Transition Purine for purine; pyrimidine for pyrimidine Transversion Purine for pyrimidine; pyrimidine for purine Insertion The addition of one additional nucleotide Deletion The removal of one nucleotide Missense Mutation Point mutation leading to a codon for a different amino acid Nonsense Mutation Point mutation leading to a stop codon Silent Mutation Mutation that doesn’t result in a change in amino acid Frameshift Mutation Insertion/deletion leading to a change in reading frame of a gene Triplet Repeat Expansion Rare mutation in which a 3 nucleotide sequence is duplicated/repeated in the genome Chromosomal Mutation Deletion Loss of a portion of a chromosome Duplication Duplication of a region of a chromosome Inversion Occurs when a region of a chromosome is removed, inverted, and reinserted into a chromosome Translocation Occurs when a portion of a chromosome is broken off and joined to a different chromosome ChadsPrep.com 37 2.4 Regulation of Gene Expression The majority of the regulation of gene expression occurs at the initiation of transcription. Regulatory proteins bind DNA and affect the ability of RNA polymerase to bind to the promoter. Prokaryotic Regulation of Gene Expression In prokaryotes control of gene expression allows the cell to respond to environmental changes. Negative Control Repressor proteins bind to operators decreasing transcription initiation. Effectors bind repressors resulting in either an increase or decrease in DNA binding. Positive Control Activator proteins bind DNA increasing transcription initiation by recruiting RNA polymerase to the promoter. Effectors bind activators resulting in either an increase or decrease in DNA binding. Trp Operon (Negative Control) Repressible Operon – Normally on but repressed by an effector The trp genes in the trp operon allow for the synthesis of tryptophan in certain bacteria. The trp operon is only induced in the absence of tryptophan. The trp repressor binds the operator preventing transcription, but only when tryptophan is bound. Tryptophan acts as a corepressor. ChadsPrep.com 38 Lac Operon Inducible Operon – normally off but induced by an effector The lac genes in the lac operon allow for the catabolism of lactose in certain bacteria. The lac operon is only induced in the presence of lactose AND the absence of glucose. In the absence of lactose, the lac operon is expressed at a very low level. Lac Repressor (Negative Control) The lac repressor binds to the operator preventing transcription. Allolactose, an isomer of lactose, is present with lactose and acts as an inducer. It binds the repressor removing it from the operator allowing transcription to occur. Glucose Repression (Positive Control) When glucose is present, the lac operon is not induced. Induction of the lac operon requires the binding of the catabolite activator protein (CAP) a.k.a. the cAMP receptor protein (CRP) at a binding site upstream of the operon. This binding only occurs when cAMP is bound to CAP/CRP. But cAMP levels are low when glucose is abundant, thus preventing induction. Glucose also prevents transport of lactose into the cell, thereby excluding allolactose too. ChadsPrep.com 39 Eukaryotic Regulation of Gene Expression Gene expression is much more complex in eukaryotes than prokaryotes and so is its regulation. Several different routes of regulation are possible. Regulation of Chromatin Structure (and Epigenetics) In eukaryotes, the DNA is packaged as chromatin, the basic functional unit of which is the nucleosome. The nucleosome is comprised of a complex of histone proteins around which the DNA is wound. The promoter of a gene may not be accessible if it is wound around a nucleosome and that gene won’t be expressed. Also, the more highly condensed the DNA in a given region, the less available it is likely to be to transcription. Genes in heterochromatin, for example, which is highly condensed are typically not expressed. Histone modification can influence the immediate chromatin structure. Histone acetylation of lysine residues promotes transcription by decondensing the chromatin structure. Alternately, histone methylation typically leads to more condensed chromatin structure and a reduction in the levels of transcription. There are activators that recruit proteins to acetylate histones in the region of a promoter (thus activating transcription), and there are repressors that recruit proteins to deacetylate histones in the region of a promoter (thus reducing transcription). DNA can also be methylated (usually on cytosine residues) which is often associated with inactive genes such as in an inactive X-chromosome (Barr body) or genes in cells in which they are not expressed. Newer research has also brought to light a role for non-coding RNAs (ncRNAs) in recruiting proteins involved in chromatin remodeling. Such non-coding RNAs have been shown to to play a role in heterochromatin formation and the aforementioned X-chromosome inactivation. These changes in chromatin structure are heritable as they can be passed on to subsequent generations of cells in mitosis. These changes which are reversible and do not involve a change in the DNA sequence are referred to as epigenetic changes. Epigenetic changes are responsible for differential gene expression in different cells in the human body. Almost all cells in the human body share a common genome, but differ in which set of genes are expressed, much of which is a matter of epigenetics. Epigenetics can be influenced by environmental factors such as diet, smoking, and infection. Changes in epigenetics have been linked to heart disease, schizophrenia, type 2 diabetes, and cancer. Abnormal levels of DNA methylation can alter gene expression. If this occurs for certain genes, the individual may be more susceptible to certain cancers. ChadsPrep.com 40 Transcriptional Regulation via Transcription Factors In eukaryotes, RNA polymerase does not bind directly to the promoter; binding is mediated through general transcription factors. These transcription factors bind to the promoter (including the TATA box specifically) and then recruit RNA polymerase II. These general transcription factors are necessary to accomplish a low, basal level of transcription. In addition to promoters there are also enhancers in the DNA to which specific transcription factors called activators bind. Their action is specific, often tissue-dependent or time- dependent, and serve to upregulate transcription above basal levels. Activators bind to these enhancers and also serve to recruit RNA polymerase. These enhancers may be upstream or downstream of the gene and are often rather distant from the gene. The DNA loops in such a way that enhancers and promoters are brought into proximity. Repressors may bind to the enhancer region thus blocking the activator or may bind the activator directly thereby decreasing its affinity for the enhancer. Some repressors have their effect by binding to silencers in the DNA instead. Steroid hormones often achieve their function by binding inducible activators resulting in their binding to specific enhancers. The steroid hormones affect the gene expression of multiple genes, and they do so as these genes have similar enhancers and are thereby affected by the same activator. This is the method of coordinated control of gene expression of related genes in eukaryotes as operons are not present as in prokaryotes. Non-steroid hormones typically cannot diffuse across cell membrane but rather bind a cell receptor at the cell surface. This results in a signal transduction pathway that ultimately activates certain transcription factors. Posttranscriptional Regulation Alternative splicing can lead to many different gene products from a single pre-mRNA, all occurring after transcription. Which exons are used is controlled by regulatory proteins. It’s been found that greater than 90% of protein-coding genes undergo alternate splicing. mRNA degradation can also affect gene expression. The addition of the 5’ CAP and poly-A tail increase the stability of the mRNA transcript. Additionally, the nucleotide sequence in the 3’ untranslated region has been found to influence mRNA stability. The shorter the half-life of the mRNA, the lower the overall gene expression. Translation initiation can also be a control point. There are regulatory proteins that can bind to the untranslated regions to block ribosomal binding and thus decrease gene expression. Protein degradation can also serve to alter gene expression. The half-life of proteins vary, but a protein can be marked by ubiquitin tagging for degradation by proteosomes. miRNA (microRNA) and siRNA (small interfering RNA) are RNA molecules of 20-25 nucleotides in length that bind to complimentary mRNA molecules. This leads to the degradation of the mRNA and/or the blocking of translation thus ‘silencing’ the gene. With siRNAs specifically, this is referred to as RNA interference (RNAi). ChadsPrep.com 41 2.5 Biotechnology Recombinant DNA – DNA that originates from more than one source Restriction Endonucleases – cut DNA at specific, often palindromic, sequences commonly leaving ‘sticky ends’ or less commonly blunt ends. DNA/Gene Cloning – a segment of DNA or a gene is incorporated into a cloning vector (such as a plasmid) using appropriate restriction enzymes and DNA ligase. The recombinant plasmid is then introduced into bacterial cells. Cells that take up the plasmid are said to be ‘transformed.’ The plasmid will typically include a marker such as antibiotic resistance that will facilitate isolating and identifying transformed cells. Gel Electrophoresis – allows for the separation of DNA fragments based upon size. A solution of a mixture of DNA fragments are placed in a well on either an agarose or polyacrylamide gel. A current is applied and negatively charged DNA migrates toward the positively charged anode. Smaller fragments travel faster and therefore farther. ChadsPrep.com 42 Polymerase Chain Reaction (PCR) - Amplification of specific DNA using specific primers and many rounds (~30) of DNA replication 1. Denaturation – the sample is heated to ~95oC to separate strands of dsDNA 2. Annealing of Primers – Appropriate primers are added and the sample is cooled to 50-70 o C 3. Elongation – the sample is heated to ~70oC where Taq polymerase synthesizes DNA cDNA (Complimentary DNA) – DNA produced from mRNA using reverse transcriptase Reverse Transcription PCR (RT-PCR) – used to amplify RNA into DNA using reverse transcriptase. It can also be done quantitatively to measure levels of gene expression. DNA Fingerprinting – Individuals are polymorphic for short tandem repeats (STRs) that are in noncoding and nonregulatory parts of the genome, and a combination of STRs can be used to identify an individual. RNA Interference (RNAi) – Allows for a temporary reduction in gene expression which is useful in the study of gene function. RNA molecules of 20-25 nucleotides in length bind to complimentary mRNA molecules either leading to their degradation or the inhibition of translation. ChadsPrep.com 43 CRISPR/Cas9 – A guide RNA can be used to direct Cas9, a nuclease, to a complimentary site where it cuts the DNA. 1. Inaccurate repair can result in insertions/deletions and loss of gene function. 2. In the presence of donor DNA, homology- directed repair incorporates the donor DNA which makes the insertion of a gene (i.e. gene editing) possible. Transgenic Organism – an organism whose genome has been altered via any method other than conventional breeding “Knockout” Mice – A cloned gene is disrupted with an antibiotic resistance gene rendering it nonfunctional. This disrupted gene is then introduced into embryonic stem cells. A double recombination event results in the normal gene being replaced with the disrupted gene. The cells are grown up in the presence of the antibiotic drug so that only cells that have incorporated the disrupted gene survive. These cells are injected into an embryo which is implanted into a female mouse resulting in chimeric mice. The chimeric mouse is bred with a normal mouse to produce some heterozygous mice. These heterozygous mice can then be bred to produce some homozygous mice. “Knockin” Mice – Similar process as seen with “knockout mice” except that a mutant allele is used instead of a nonfunctional variant. The change in phenotype then reveals the effects of the mutant allele. Fluorescent in situ Hybridization (FISH) – Fluorescently tagged single-stranded DNA probes that are complimentary to a specific DNA sequence can be used to identify the presence of specific DNA elements or alleles for gene-mapping. ChadsPrep.com 44 DNA Microarrays (a.k.a. Gene Chips) – can be used to detect the presence of specific DNA sequences or to measure differential expression of large numbers of genes in a sample. In measuring the differential expression of genes, specific single-stranded DNA probes corresponding to the genes being assayed are immobilized in defined locations on a chip. Collected RNAs are converted into cDNA via reverse transcriptase, tagged with a fluorescent marker, and then allowed to hybridize to the DNA probes on the chip. Fluorescence can then be used as a measure of gene expression. RNA-seq – can be used to detect and quantify all RNAs expressed in a sample (not just the ones for which DNA probes have been prepared as with DNA microarrays). Collected RNAs are converted into cDNA via reverse transcriptase and then sequenced using Next-Generation Sequencing (NGS). While having the advantage of assaying ALL RNAs, it is more expensive than DNA microarrays. Enzyme-Linked Immunosorbent Assay (ELISA) – Allows for the quick detection of a specific antigen which is helpful in the diagnosis of various infections. An antibody specific to the antigen with a linked enzyme is used. If antigen is present it will bind and the addition of the enzyme’s substrate results in a signal and a positive test for the antigen. ChadsPrep.com 45 2.6 Genomics Genomics – the study of all the DNA (including all genes) in an organism Genome – the sum of all DNA in a cell/organism Bioinformatics – the application of computational analysis to interpret biological data Metagenomics – the study of all genetic material from an environmental sample Gene Annotation – the identification of protein-encoding genes (open reading frames—ORF) in a genome Genome Mapping Gene Mapping – the relative positions of various genetic markers (genes, restriction sites, ESTs, etc.) within a genome Genetic Map – map of the genome giving relative positions of genetic markers based upon recombination frequencies (measured in centimorgans—cM) Physical Map – map of the genome giving absolute positions of genetic markers in base pairs Expressed Sequence Tag (EST) – a small sequence of cDNA that is mapped onto the genome DNA Sequencing Shotgun Sequencing – DNA is fragmented and overlapping fragments are sequenced. The regions of overlap allow the sequence of the entire genome to be determined. Sanger Sequencing (Dideoxy Chain Termination Sequencing) - DNA is sequenced via replication with fluorescently labeled dideoxy nucleotides mixed with standard nucleotides. Each dideoxy nucleotide is labeled with a different color fluorophore. As the DNA is replicated there are some fragments that will terminate at every single base labeling that fragment with the chain-terminating nucleotide. Gel electrophoresis is used to separate/resolve the fragments with each being scanned for fluorescence revealing the nucleotide at that location. Next Generation Sequencing (NGS) – DNA is fragmented, amplified, and then sequenced in a massively parallel way. The DNA fragments are sequenced via replication with 4 unique fluorescently labeled nucleotides. Replication is carried out one nucleotide at a time with scanning for fluorescence performed in between each successive nucleotide to determine which nucleotide was incorporated each step along the way. ChadsPrep.com 46 Human Genome COMPARISON OF GENOMES Prokaryotes Eukaryotes Humans Size of Genome 0 - 6x10 bp 6 12x10 - 149x10 bp 6 9 3x109bp # of Genes 1,500 – 7,500 5,000 – 40,000 ~20,000 HUMAN GENOME COMPOSITION Protein-Encoding Genes (Exons) 1.5% Introns 24% Gene Regulatory Sequences 5% Unique Noncoding DNA 15% Transposable Elements 45% Number of Pseudogenes 14,000+ (0.5%) Transposons – mobile genetic elements that can move to different locations in the genome “Cut and Paste” – the transposon is excised and reinserts in the genome “Copy and Paste” – the transposon is duplicated; the new copy inserts in a new location Retrotransposons – a mobile genetic element which uses reverse transcriptase to convert an RNA transcript into cDNA which then inserts in a new location Multigene Families – clusters of similar genes that are likely the result of duplication examples include the histones, rRNA, and the globin gene family clusters Polyploidy – potentially the result of nondisjunction; this could result in an extra copy of a chromosome that is free to mutate and gain new function Single Nucleotide Polymorphisms (SNPs) – loci in the genome where variation is found in at least 1% of representatives of a species Copy Number Variants (CNVs) – loci where some individuals have 1 or multiple copies of a gene Comparative Genomics – the comparison of multiple genomes to identify possible functions of genes as well as relatedness of organisms Functional Genomics – Genomics investigating the relationship between genotype & phenotype Transcriptome – the sum of all mRNA expressed by a cell/organism Proteome – the sum of all proteins expressed by a cell/organism ChadsPrep.com 47 2.7 Developmental Mechanisms Cell Division Rapid cell division of the zygote follows fertilization. Cell Differentiation Cell differentiation occurs when a cell has become a distinct cell type. It is the result of differential gene expression that are in part controlled by epigenetic changes. Cell Determination – The ‘choosing’ of a particular fate for cell type even though it isn’t yet apparent. Differentiation is the result of determination. There are 2 major controlling factors to cell determination: 1. Cytoplasmic Determinants – Maternal mRNA and proteins are present and unevenly distributed in the ovum (and zygote). Subsequent cell division will result in cells with varying concentrations of these cytoplasmic determinants that can ultimately lead to gradients of these determinants in the developing embryo. The cytoplasmic determinants act as transcription factors and varying concentrations will therefore lead to differential gene expression and play a role in cell differentiation. 2. Induction – There are signal transduction pathways present allowing changes in gene expression to be caused by neighboring embryonic cells that can contribute to cell differentiation. Totipotent – Cells that can give rise to any cell type including extraembryonic cells. Pluripotent – Cells that can give rise to any cell type except extraembryonic cells. Multipotent – Stem cells that can give rise to a limited number of cell types. Unipotent – Stem cells that can only give rise to a single cell type. Embryonic Stem Cells – pluripotent cells derived from a mammalian blastocyst ChadsPrep.com 48 Pattern Formation and Morphogenesis 1. Morphogen gradients establish the major body axes. 2. The sequential activation of genes leads to body segmentation. 3. Segment identity is controlled by homeotic genes (including Hox genes). Hox genes (i.e. homeobox-containing genes) are transcription factors that affect cell behavior associated with organ morphogenesis. The homeobox is a 180nt (60aa) DNA-binding domain. Homeosis – Occurs when one body segment takes on the identity of another. Morphogenesis – The production of ordered form and structure. Morphogenesis is the result of regulation of all of the following: 1. Cell Division – The number, timing and orientation of cell division is highly regulated. Additionally, unequal cytokinesis plays a role in establishing particular cleavage patterns. 2. Cell Size/Shape – Large changes in cell size and shape may accompany cell differentiation such as in nerve cells of the spinal chord. 3. Cell Migration – Differentiating cells need to migrate to their proper location in the developing embryo. The ability to modulate adhesion both to other cells as well as to the extracellular matrix is essential. Cadherins – Transmembrane proteins that mediate Ca2+-dependent homophilic binding. Integrins – Transmembrane proteins used to bind the cytoskeleton to the extracellular matrix. Binding can serve as an anchor but can also activate signal transduction pathways leading to growth of the cytoskeleton or the activation of gene expression. 4. Cell Death – Apoptosis is a normal part of embryo development and may play a role in the following: a. Sculpts form b. Destruction of vestigial organs c. Separation of tissue layers d. Creation of lumens ChadsPrep.com 49 Embryogenesis Fertilization Sperm contributes a nucleus only. The ovum contributes a nucleus, organelles, and cytoplasm. Fertilization usually takes place in the fallopian tubes during the 4-5 day journey to the uterus. 1. Acrosomal Reaction - Degradative enzymes from the sperm’s acrosome disperse the corona radiata and break down the zona pellucida allowing penetration. Penetration also results in the influx of Ca2+. 2. Cortical Reaction – Ca2+ signals the release of the contents of cortical granules into the extracellular matrix. This catalyzes the hardening of the egg making it impenetrable to additional sperm (blocking polyspermy). 3. Activation - Ca2+ also activates the ovum. Upregulation of cellular respiration and protein synthesis. The ovum completes meiosis II. The two nuclei fuse restoring the diploid state. The fertilized ovum is now called a zygote. ChadsPrep.com 50 Cleavage During cleavage cell division occurs with little cell growth. Cleavage includes 2-cell, 4-cell, and 8-cell stages. Individual cells at this stage are referred to as blastomeres. In humans blastomeres up to the 4-cell stage are totipotent. Blastula (a.k.a. Blastocyst in mammals) Formation A hollow ball of ~100 cells that will embed in the endometrium (implantation). trophoblast - outer layer of cells that becomes the chorion/placenta (secretes hCG) -chorionic villi pierce the vascular endometrium inner cell mass – mass of cells that will become the growing embryo blastocoel - hollow fluid-filled cavity Gastrulation The blastocyst folds in on itself (invagination) forming the three germ layers setting the stage for organogenesis. Cadherins and integrins are crucial for gastrulation. The notochord forms in chordates and ultimately will parts of the vertebral discs. It also plays a role in signaling/coordinating formation of the neural plate/tube (neurulation). Archenteron – central cavity destined to become the gut GERM LAYERS ECTODERM Epidermis, nervous system, sense organs Dermis, muscle, bone, connective tissue, cardiovascular system, MESODERM lymphatic system, urinary system, reproductive system ENDODERM Respiratory & digestive epithelia, digestive system, bladder Neurulation The central nervous system is formed from the ectoderm. The neural plate folds to become the neural tube (ultimately becoming the brain and spinal chord). Neural crest cells pinch off from the edges of the neural tube and migrate and differentiate to become the peripheral nervous system and a variety of other tissues. ChadsPrep.com 51