ANTHR 2750 Lecture 2

• Obtain genomic DNA sample
• 2. Sequence genomic DNA
• 3. Assemble sequences in order
• 4. Annotate sequence

• Hierarchical sequencing:
• Genomic DNA is cut into pieces of about 150 Mb and inserted into BAC vectors, transformed into E coli where they are replicated and stored. The BAC inserts are isolated and mapped to determine the order of each cloned 150 Mb fragment.

• Shotgun Sequencing:
• Randomly shears genomic DNA into small pieces which are cloned into plasmids and sequenced on both strands, thus eliminating the BAC step from the HGP’s apporach and deeming it more efficient.

• Genome assembly.
• Based on finding regions of overlap between individual sequencing fragments.
• This is a difficult problem for complex genomes.

Identifying what part of DNA corresponds to gene.

Celera (private effort)

• Compare to known genes already described and sequence;. Through expressed sequence tags (EST) and randomly produced mRNA to infer genes
• Predict genes computationally: Using computer predictions to determine shape of protein and infer function.

• Euchromatic: 93% of DNA, contains most of the genes in your genome, 99% has been sequenced.
• Heterochromatic: 7% of your DNA, highly repetitive. Some parts are structural, contains centromeres and telomeres. Genes are quite sparse. Very difficult to sequence due to repetition and thus largely unexplored.

• 93% of DNA,
• contains most of the genes in your genome
• 99% has been sequenced.
• 30,000 genes. (many fewer than expected)
• 50% have unknown function
• Less than 2% of the genome is a gene
• 98% is “junk” DNA. Many repeated sequences and transposable elements with unknown functions.
• 98% does not code for genes. The function of non-coding elements is unknown, but potentially very important.
• 50% of repeated sequences are transposable elements.

Sequences of DNA that can move around to different positions within the genome of a single cell, a process called transposition.

When removing introns, processes such as skipping exons, intron retention can occur, leading to varying gene expression.

• Any duplication of a region of DNA that contains a gene.
• It may occur as an error during homologous recombination, a retrotransposition event, or duplication of an entire chromosome.

They occupy 50% of our genome.

Barbara McClintock suspected the existence of transposons in 1940. The existence of transposable elements proven experimentally in the 1970s. Received the Nobel Prize in 1983. Cornell University

• LINEs (long interspersed nuclear elements) retroposons
• SINEs (short interspersed nuclear elements) retroposons
• DNA transposons
• Retroviruses and retrovirus-like LTR (long terminal repeat) retrotransposons.

• LINE retroposons and retrovirus-like retrotransposons code for reverse transcriptase. Go through the intermediate RNA phase.
• LINE1 is the most common.

• Acquire reverse transcriptase from other elements.
• Alu helps the process of duplicate regions.

Code for transposase and insert double stranded DNA into host genome.

• Alu element tends to insert itself into G C rich regions. (G C tends to be more frequent in coding areas)
• LINE1 element tends to insert itself into G C poor regions.

• Activity (insertion) of Alu peaked at 7% divergence (based on molecular clock, substitution from consensus sequences), corresponding to 40 Myr, then dropped. This is when it evolved. We can see Alu in chimpanzees as well.
• Activity of DNA transposons and LTR retrotransposons started diminishing earlier.

• Some are inversions or deleterious, thus causing disease etc.
• Can cause genome instability. (cancer)
• Although it might be rare, RE can also contribute to coding in regulatory regions of functional genes.

Gene duplication and mutation.

• Single gene duplication (including retro-transposition)
• Segmental gene duplication (including whole chromosome duplication)
• Whole genome duplication

Two genes are homologous if they have a common ancestor.

Two genes are orthologous if they diverged following a speciation event.

Two genes are paralogous if they diverged following a duplication event.

500 genes in humans and 1400 genes in mice.

• More copies in the genome of populations that consume high starch to produce more amylase.
• High copy number variation gives us hints about evolutionary history.

• Genetic duplication leads to genetic novelties
• Nonfunctionalization (pseudogenes)
• Subfunctionalization
• Neofunctionalization (evolution of new function, beta version of the function)

One theory formalized in 1970 that the complexity of the vertebrate genomes originated by means of genome duplication at the base of the vertebrate lineage. Since then, the theory has remained both popular and controversial.

• 1. The Human Genome Project was thought up by Charles DeLisi in 1985 with the aim of sequencing the DNA of the human genome and determining all sequences coding for genes. 
• 2. The project officially began in 1990. This was also when the Ethical, Legal and Social Implications Program was created. 
• 3. Craig Venter, an NIH scientist believed that he could cut costs and time by using "shotgun sequencing" rather than "hierarchical sequencing" and founded Celera. 
• 4. Both projects published a working draft in 2000. 
• 5. In 2003, the initial project was completed.

• 1. Targeted medication with minimal side effects. 
• 2. More effective vaccines made of DNA and RNA reducing risks of vaccines. 
• 3. More accurate dosages based on specific metabolisms. 
• 5. Early diagnosis. 
• 6. The rescue of drugs that may work for some people and not for others.

• 1. Denial of insurance. 
• 2. Denial of employment. 
• 3. Increases inequality in access to these technologies leading to genetic aristocracy. 
• 4. Determinism element associated with disease etc. leading to lessened efforts to improve environmental conditions. 
• 5. Privacy issues with access to DNA data.