Difference gel electrophresis

Samples are dyed different colours, mixed, and separated by 2D gel electrophoresis. Determines relative abundance in each sample, and identifies protein with significant differences.

Isolate DNA from cancer cells, fragment DNA into small pieces. Transfect DNA into 3T3 cells, each cell takes up a different fragment. Grow cells and identify foci, groups of cells growing in clumps. Isolate the human DNA fragment, with human repetitive DNA.

1. Transfection of human bladder carcinoma DNA identified as a sequence that was identical to H-ras oncogene from Harvey rat sarcoma virus.

2. Transfection of DAN from normal human cells transformed by treatment with a chemical carinogen identified as a sequence related to the Ki-ras oncogene from Kirsten rat sarcoma retrovirus.

A form of next generation sequencing. Reads are about 300 - 500 nucleotides, but one sequencing reaction can produce millions of reads. Reads one gigabase (1/3 of the human genome) in a single day. It is much cheaper than Sanger sequencing: $0.02/1000 bases.

1. Generation of a single-stranded template DNA libary. DNA is fragmented and denatured, and adaptors are added to each end

2. EMulsion-based clonal amplification of the library. Each DNA fragment is bound to a bead, encapsulated in emulsion. DNA is amplified by PCR within the micelle, beads are then loaded into a picotiter plate

3. Pyrosequencing

4. Data is analyzed using bioinformatics tools. The order of nucleotides provided and the amount of light emitted by pryosequencing gives the sequence.

Reaction mixture: DNA polymerase, nucleotides, luciferase, apyrase, ATP sulfurylase, luciferin, and APS

"Falling away"

Programmed cell death. Caspases attack proteins in the cell, chromatin becomes fragmented. The cell is usually engulfed by a phagocyte and destroyed. Cells that have potential to become cancerous often undergo apoptosis. The molecular and morphological process leading to controlled cellular self-destruction. Sculpts the developting embryo by removing transient structures, including formation of digits. All physiological cell death occurs by apoptosis.

1. Reduction in nuclear size, condensation of chromatin at nuclear periphery and detachment from surrounding cells.

2. Cell shrinkage, blebbing of cell membrane producing apoptotic bodies.

3. Apoptotic bodies phagocytosed by macrophages.

Mouse ear cress

A plant model organism with a sequence genome; sequenced in 2000. There are detailed physical maps available for its genome. The mitochondrial genome is 366,924 bp, with 57 genes. The chloroplast genome is 154,478 bp, with 129 genes. Its genome is 119,186,494 bp with 28,152 genes, for a density of 4,200 bp/gene. There are about 1012 protein domains. Insertion sites have been mapped in hundreds of thousands of transgenic plants using reverse genetics with T-DNA. Seed stocks are available to researchers on request.

Concluded that the evolution of mammalian chromosomes involved three classes of conserved synteny:

1. Conservation of entire chromosomes

2. Conservation of large segments of chromosomes

3. Joining of segments of different chromosomes to produce new synteny

Basic Local Alignment Search Tool

Used to search GenBank for homologues of genes. Amino acid sequence alignments can identify proteins with related functions.

Breast cancer 1

Early onset genes encodes a protein involved in DNA repair.

CCAAT

A basal promoter sequence. Binds CAAT binding protein transcription factors, such as NF1 and CTF.

Roundworm

A see-through nematode. A model organism. Its genome was sequenced in 1998. There are detailed physical maps available for its genome. The mitochondrial genome is 13,794 bp, with 12 genes. The genome is 100,291,841 bp with 20,516 genes, for a density of 4,9000 bp/gene. Has 1090 cells. Cell fate is completely mapped. RNAi can be performed by microinjecting dsRNA, soaking them in media containing dsRNA, or fed bacteria synthesizing dsRNA. RNAi gave new insight into the function of over 400 genes. Used as a model system for the analysis of diseases of protein mis-folding. Used to screen for genetic and chemical modifiers of Huntington's disease.

Cysteinyl aspartate-specific proteinase

Proteolytic enzymes that remove small aprts of other proteins, inactivating them. Cysteine proteases responsible for the ordered dismantling of cellular structures during apoptosis. Cleave after aspartic acid (D) residues. Present in an inactive pro-form in healthy cells. Attack many types of protiens, including lamins and the cytoskeleton. Degrade specific proteins that lead to death of the cell. There are two protease subunits, large and small, a cleavage site, and a prodomain with a death fold. Includes ICE sub-family, and CED-3 sub-family. Includes effector and initiator caspases.

Complementary DNA

Complementary to mRNA. Requires reverse transcriptase to synthesize. DNA molecules created from RNA remplates. The first strand of DNA is created by reverse transcriptase, complementary to the mRNA. Created using oligo(dT) primers that anneal to the poly(A) tail of the mRNA, or gene-specific primers, often annealing to non-coding regions near the poly(A) tail at the 3' end. Gene-specific primers are used for reverse transcriptase-PCR amplification of RNAs. Double stranded DNA molecules are synthesized usign ribonuclease H, DNA polymerase I, and DNA ligase.

Cystic fibrosis transmembrane conductance regulator protein

Encoded by CFTR. A membrane channel protein through which ions pass. 1480 amino acids. Some evidence suggests CFTR has a role regulating lipid metabolism and transport. Mutant CFTR caused by a ΔF508 mutation is non-functional, and is tagged by ubiquitin for degradation. As a result, salt accumulates in epithelial cells and mucous builds up on the surface of the cell.

2'3'-didoexyribonucleoside triphosphates

Chain terminators used in the Sanger sequencing protocol. Terminates DNA synthesis when incorporated because they have a 3'-H rather than 3'-OH. Labelled with different fluorescent dyes: ddATP is green, ddCTP is blue, ddGTP is black, and ddTTP is red.

Linkage

The presence of genes/markers on the same chromosome.

There are two types:

1. Interact with components of basal transcriptional machinery. General transcription factors and mediator complex activate RNA polymerase II.

2. Interact with chromatin to create regions of nucleosome-free DNA. Includes histone modification complex and chromatin remodeling complex.

Combined DNA Index System

Used by the FBI in forensics. 13 STRs are used. The probability of two individuals having identical fingerprints at all 13 loci is 1 in 5.75 trillion. For each "allele", there is a different number of repeat of the same STR. Loci amplified, dyed with fluorescent dye, and separated with gel electrophoresis. Up to nine loci may be done at a time.

A set of contigs. Made using restriction enzyme mapping, DNA sequencing, and STS mapping. Fragments are ordered to reflect the sequence in the genome; compare RE digest patterns for each of the fragments. Each portion of the genomic DNA will be present in sevreal different plasmids since DNA is randomly fragmented. Each clone must be cut with several restriction enzymes; the size of each fragment produced in single and combination digests is used to construct a map of each clone. Computer software is used to order the clones. From a YAC clone, two libraries are produced, each from different restriction enzymes. Southern blots are prepared for both, and used to see which fragments align with each other, to create a contig map.

1. Begin as YAC clones, subcloned into smaller fragments in general purpose cloning vectors

2. Create restriction maps or DNA sequences of each clone

3. Contigs identified by overlapping restriction map patterns and sequences

 Clustered regularly interspaced short palindrome repeats

Short stretches of phage DNA separated by repeated short bacterial DNA sequences. Transcribed into pre-crRNA.

Zebra fish

Its genome is 1,571,018,465 bp, with 23,524 genes, for a density of 66,800 bp/gene.

Bubble Boy

Born in 1971 with SCID. Lived in a specially sterile enclosure, at first in a hospital, then at home. Everything in the enclosure had to be sterile to prevent infections. Received a bone marrow transplant from his sister at age 12, but died 15 days later from Burkitt's lymphoma caused by a latent Epstein-Barr virus in his sister's bone marrow.

DNA fingerprint

Specific banding patterns produced by DNA fingerprinting. Individual bands of DNA fingerprints represent different alleles of VNTR/STR loci. The probability of two individuals (not identical twins) having the same DNA fingerprint decreases the more polymorphisms are used. Used for CODIS, used in paternity tests and forensics. Routinely used to identify and/or distinguish individuals. 

DNA fingerprinting

PCR is used to amplify DNA found in very small tissue samples. Detection of VNTR or STR polymorphisms by PCR and Southern blotting/capillary gel electrophoresis. The use of DNA sequences in personailty identity cases. A valuable tool in cases of uncertain identity including paternity cases, crimes, or disasters. It was used to identify body parts recovered in teh debris after the collapse of the Twin Towers on 9/11.

Cheks for ssDNA, ds breaks, or DNA modifications. Arrests cell cycle, repairs damage, then resumes. If damage is nonrepairable, the cell undergoes apoptosis. Tumour suppressor genes may have loss of expression or express mutant cell cycle inhibitor p53, or pRNB.

p53 → p21 (CDK1) --| CDK2/cyclin E/A

Proposed six hallmarks to the pathways leading to malignant cancer:

1. Cancer cells acquire self-sufficiency in signalling processes that stimulate division and growth

2. Cancer cells are abnormally insensitive to signals that inhibit growth

3. Cancer cells can evade programmed cell death

4. Cancer cells can acquire limitless replicative potential

5. Cancer cells develop ways to nourish themselves

6. Cancer cells acquire the ability to invade other tissues and colonize them

Executioner caspase

Cleaves specific cellular proteins, leading to cellular inactivation. Includes caspases 3, 6, and 7.

"Above'

A heritable state other than the actual sequence of the gene regulates the gene's expression. Includes chromatin organization, acetylation of histones, and methylation of histones or nucleotides.

"True"

Lightly stainign chromatin. Most eukaryotic genes are located in euchromatin. Generally active.

Short cDNA sequences used as hybridization probes to anchor physical maps to genetic maps and cytological maps. Usually short, less than 1000 bp, single-pass sequence reads from mRNA. Typically produced in large batches. Represents a snapshot of genes expressed in a cell at a certain time. They are tags, some coding, others not, of expression for a cDNA library. Represent portions of cDNAs generated by sequencing cDNA fragments. Used for gene mapping, studying transcripts and SNPs, discovering putative gene products, comparing gene expression patterns, and as probes for microarrays or gene chips.

To generate an EST library:

1. Isolate total mRNA, using an oligo dT column to trap all poly(A) containing mRNAs

2. Add reverse transcriptase and oligo dT primers, to generate a single-stranded cDNA for each mRNA

3. Convert ss cDNA into ds cDNA (with DNA polymerase)

4. Generate a ds cDNA library by cloning products into a vector

5. Select specific ESTs as markers

Fragile X Mental Retardation Protein

An RNA-binding protein, playing a role in transporting mRNA molecules, or in regulating their translation. In Fragile X syndrome patients, it builds up in the dendrites of neurons.

Have GTPase activity. Conformation is altered by GDP/GTP binding. GDP bound is inactive. GTP bound is active.

GTP → GDP

Requires nutrient/growth factor independent synthesis of late G1 cyclin (cyclin E). Checks if cell division is compatible with function of tissue and well-being of the organism. Oncogenes may overexpress or produce hyperactive signalling molecules or transcription factors controlling the expression of cyclin D.

Growth factors → cyclin D --| pRB --| E2F → cyclin E

Red jungle fowl

An ancestor of domestic chickens. An organism with a sequenced genome.

GGGCGG

A basal promoter sequence. Binds SP1 basal transcription factor.

ncbi.nlm.nih.gov/genbank

Proposed by Walter Goad in 1979. Contains all sequenced genes. Can be used to design special primers. Maintained by NCBI. By 2011, it contained over 117 billion bp of sequenced DNA. It became part of the International Nucleotide Sequecne Databse Collaboration.

Based on relative positions of genes, RFLPs, STRs, VNTRs, and SNPs, based on lineage studies. Genetic techniques are used to construct maps showing the relative positions of genes and other sequence features in the genome. Cross-breeding, or pedigree analysis is used to determine recombination frequencies. Map distance is the frequency of recombination between two loci on the same chromosome, due to crossing over. Loci that are farther apart have greater chance of crossing over. Good for mapping the gene order and relative distance of genes. Distances do not always correlated with phsycial distances.

1 cM = about 1 mb = 1 million bp

Diagnostics

An application of DNA microarrays. Labelled genomic DNA of a gene is hybridized into an array of alleles of gene. A one-nucleotide difference in a SNP can prevent hybridization.

Heritable gene therapy

A type of gene therapy. Performed experimentally in mice and other animals, never in humans. Moral and ethical considerations are involved.

Allows visualizaion of proteins in real time. Studies the subcellular localization of proteins by cloning GFP "in frame" with the open reading frame of a specific gene, and viewing by UV fluorescence microscopy. Many colours can be used at once, for different proteins.

1. Clone the open reading frame of the gene of interest, "in frame" with the GFP open reading frame

2. Transfect the plasmids into cells

3. Visualize in real time with fluorescence microscopy

Roundup Ready

An EPSP synthase gene resistant to glyphosate is transferred into crop plants using Ti plasmid mediated transformation. The crops can be sprayed with glyphosate to kill weeds, without affecting growth of the crop. It is not allowed to save Roundup Ready seeds from a crop to plant them next year.

(α/β globins)

Store oxygen in blood.

"Different"

Darkly staining chromatin. Represses gene function, perhaps because it is condensed into a form that is not accessible to transcriptional machinery. Rich in repetitive DNA, mostly located in centromeres or telomeres. Generally inactive.

Histone acetylases

A mechanism of chromatin remodeling. Enzymes that transfer acetyl groups to lysine at specific positions in histones of nucleosomes. This is associated with increased gene expression. Loosens DNA-histone associations in nucleosomes, and "opens" the chromatin. Acetylation of histones is correlated with increased gene expression. Creates open chromatin to allow transcription factor binding to promoters/enhancers.

Humans

The genome is sequenced. There are detailed physical maps available for the genome. The mitochondrial genome is 16,571 bp, with 37 genes. The genome is 2,851,330,913 bp with 22,287 genes, for a density of 127,900 bp/gene. There are about 1262 protein domains.

huntingtin

Used to be called IT15 (Interesting Transcript 15)

The mutation is called mhtt. The gene responsible for HUntington's disease. Located at 4p16.3. Being homozygous mhtt leads to more rapid onset of symptoms. One of the first human genes to be shown to be linked to an RFLP. Spans abotu 210 kb near the end of the short end of chromosome 4. Healthy individuals have 11 - 34 copies of the CAG repeat. Individuals with Huntington's disease have 42 - 100 copies, and age of onset is negatively correlated with number copies. Number of copies can expand and contract between generations. The gene encodes for HTT.

Huntingtin protein

It is 3144 amino acids in length, with a polyglutamine tract. Important for transcription, cell signalling, and transport. The mutant form of the protein has an excessive polyglutamine region that causes inability to fold properly, leading to accumulation of aggregates of huntingtin protein in brain cells, loss of neurotransmission, and atrophy of neuronal tissues.

Human Genome Sequencing Project Consortium

Established in 1993 by Francis Collins to coordinate the efforts of human geneticists around the world. Used chromosome walking to sequence.

White-cheeked gibbon

Comparison with human chromosomes revealed at least 21 translocations that occurred between our two species from our common ancestor.

Insulin-like growth factor

A gene that has gene imprinting. Methylated in the female germ line, and not in the male germ line. The copy inherited from the father is expressed. The copy inherited from the mother is methylated and not expressed.

Detects where in the cell a protein is localized.

1. Grow cells on a glass slide

2. Fix cells with paraformaldehyde

3. Block with a protein solution

4. Incubate with a primary antibody, which detects a protein of interest

5. Wash to remove unbound antibody

6. Incubate with conjugated secondary antibody, which detects the primary antibody

7. Wash

8. Analyze with a fluorescence microscope

A method of producing transgenic animals. DNA is injected into the nucleus of zygotes; DNA integration occurs during early development, and not all germ cells have the transgene. G₀ offspring are chimeras, and are mated to produce G₁, which are screened for the presence of the transgene.

1. Remove the eggs from the female mouse, and fertilize in vitro, producing a diploid fertilized egg.

2. Microinject DNA into the nucleus of the zygote

3. Implant the zygote into a pregnant mouse

4. Screen offspring for transgenic mice

A cytokinine receptor signal.

1. Interferon (IFN) binds to a cell surface receptor

2. IFN binding causes receptor dimerization

3. The Jak kinase associated with the intracellular domain of the receptor becomes activated by phosphorylation

4. Activated Jak kinase phosphorylates tyrosine residues on the intracellular domain of the receptor

5. PHosphorylated tyrosine residues bind to STAT transcription factors

6. Jak phosphorylates the bound STATs

7. Phosphorylated STAT dimers enter the nucleus, bind to specific DNA elements, and activate transcription of downstream genes