Genes - Human Genome Project took 13 years and 3 billion dollars. It took less time than anticipated because one gene area can code for more than 1 protein due to the relationship between introns and exons. - Now takes 24 hours and about $1000 for a personal genome. - Approximately 400 genes can mutate to produce an abnormal protein that can cause cancer. These proteins can override the “locked door” between the G1 and S (synthesis) phase of interphase, causing DNA replication, and therefore mitosis, when not needed. - Only 1% of our genome (20,000 coding genes) contains genes that make proteins. (We make over 100,000 proteins from the exons within these genes.) The other 99% is non-coding DNA used to control DNA, produce RNA, etc. - We have 3 billion nucleotide pairs, HIV only has 9,749 nucleotide pairs and 9 coding genes.

The “non-coding” DNA (about 500 genes) may be used to make RNA: 1) small noncoding RNAs (sncRNAs) of 20 to 200 nucleotides. These include: rRNA, tRNA, and microRNA (miRNA or miRs) 2) long non-coding RNAs (lncRNAs) of 200 to 1000’s of nucleotides. A special category of these, long intergenic non-coding RNAs (lincRNA) control embryonic stem cells! There are about 80 trillion cells in the adult human body. These go through replication and transcription as needed. There are about 400 oncogenes and tumor suppressor genes (in every cell with a nucleus). 80 trillion cells replicating/transcribing DNA with 400 potentially cancer causing genes in every nucleus!!!! Transcription factors can affect the promoter region of a gene, turning it on or off. MicroRNA (miRs are about 22 nucleotides) turn genes OFF. They control over 50% of coding DNA.

70% of our DNA is still NOT fully understood; “Junk DNA” or the “dark matter” of DNA. The 20,000 coding genes, making more than 100,000 proteins have to turn on/off at the correct time in all 80 trillion cells in our bodies. Transcription factors influence whether or not a gene is on (positive regulation) or off (negative gene control), often by attaching to the promoter region (5’ end) of the DNA. microRNA (miRs) is a negative regulator (turns off coding DNA) which controls over 50% of the coding DNA. However, these work at the 3’ (terminator region) end of the gene. When these mutate, we get ONCOmiRs which cause cancer. But directly influencing DNA is not the only way to regulate genes since DNA never acts alone, it is always complexed with histone proteins.

When chromatin (DNA plus histone complex) is open (loose), transcription can occur because promoter regions are accessible. When chromatin is tightly coiled, no access to promoters so no transcription. Methyl groups (CH 3 ) attach to cytosine in DNA (often at promoter region), coiling the DNA/histone complex, turning the gene off. Loss of methyl groups turns genes back on. Methylation of a tumor suppressor gene can cause cancer. Acetyl groups (COOH) that attach to the amino acid lysine in the histone proteins, turn genes “on.” If removed, genes turn “off.” This is done by uncoiling/coiling of chromatin. (Histone acetylase adds acetyl groups. Histone deacetylase removes acetyl groups.) Acetylation of an oncogene can cause cancer. Even parents’ diets can influence gene expression. Ex: Dads on high fat diets are more likely to have daughters with diabetes. Hence, the epigenome can readily change even though the actual genome is very stable..

Condensed chromatin on packed histones (lower middle) can’t be accessed for transcription/replication but loose histones can be (upper middle).

Why do we have such on/off controls? If we didn’t have the ability to turn on or off genes, how would a liver cell know it was a liver cell and not a spleen cell? Remember all cells have the same DNA. What the cell eventually becomes is based on which genes are turned on or off. Embryonic stem cells can become anything.

While the genome is quite stable, the epigenome is not. Transcription factors, miRs, etc. can affect it as can protein activation. Our protein synthesis/use can be controlled via phosphorylation. Using a kinase, such as tyrosine kinase, an inactive protein can become activated by adding a phosphate (from ATP). A protein can be deactivated by removing a phosphate using phophatase. There are over 500 human kinases affecting protein activity! This interaction between kinases and phosphatase allows a protein to hang around and be used as necessary, without having to go through transcription and translation again each time that protein is needed. Kinases can also affect cell mitosis by activating cyclin proteins. Cyclin genes must be turned “on” at the right time to regulate mitosis. Oncogenes produce abnormal proteins that turn on the cyclin genes at the wrong time. Normally, a cascade of events must occur in a specific order for the cell to go from the G1 phase of interphase to the S (synthesis) phase (when DNA is replicated because a signal to divide has been received).

Think of it as a series of locks preventing the cell from going from G1 to the S phase which would trigger mitosis. The first few “locks” are on the cell membrane, some more in the cytoplasm, and the last few in the nucleus. Normal cell signal transduction for appropriate mitosis involves about 10 “locks” changing from locked to unlocked. Usually, the correct protein “unlocks” the first signal for mitosis at an area on the cell membrane. Say there is a cut in the skin, a protein unlocks the site on neighboring skin cells. A cascade of reactions occurs until all locks are undone and the cell moves from step G1 in interphase to the S phase so that new skin cells can repair the gap caused by the cut. This should be an orderly process, always starting at the appropriate site. However, oncogene proteins can step in at various lock locations starting the cascade of events that triggers the cell to move from G1 to S at the wrong times. Once any lock along the path is undone, the rest in the sequence open – like dominoes falling – turning on the cyclin genes for mitosis. Tumors or unregulated cell mitosis results.

Since phosphorylation at these locks is key to opening them, some drugs, such as Gleevac, replace ATP at one of the locks involved (in this case,for a specific leukemia, hence no phosphorylation (PO4 comes from ATP).. ONCOGENE (ONC): a) An Oncogene makes a protein that initiates Cell Cycle & Mitosis at the wrong time: Cell Signal Transduction System Oncoproteins b) Acts as Dominant Trait, one allele mutates, ‘Gain of Function’ mutation c) May be Carried by and Expressed with RNA Viruses d) Usually NOT Inherited – Sporadic e) Proto-oncogene (normal gene) is Activated (mutates) to become Oncogene which produces the Oncoprotein f) Oncoprotein turns on the cyclin genes at the wrong time = cancer