Biology: Basic Genetics

Genetics focuses on the physical genome, or genomic nucleotide sequences of species.

Epigenetics however, is a term used to describe changes that are made around the genome that do not alter the actual nucleotide sequence. These epigenetic chemical markers and histone modifications can be passed down from one generation to the next.

• Gene - nucleotide sequence that can code for a certain product or set of products, depending on factors such as alternative splicing and protein mofidication. 

Order from DNA to Chromosomes:

Double stranded DNA --> DNA wrapped around a core of 8 histones (globular proteins) called Nucleosome --> Chromatin --> Chromosome (via coiling of the chromatin within supercoiling)

Histones have basic functional groups that give these proteins a net positive charge at the normal pH of the cell. This is to attract negatively charged DNA and assist with the wrapping process.

Important: Cellular machinery that "reads" genetic codes can only act on chromatin that is uncoiled. The structure of chromatin is hence influenced via epigenetic regulation.
     -Chromatin that is tightly condensed is called heterochromatin.
     -Chromatin that is not condensed at all is called euchromatin.

-In animals, DNA is only found in the nucleus and the mitochondria.

One type of epigenetic regulation is DNA methylation, which involves the addition of an extra methyl group to particular cytosine nucleotides. This causes the DNA to be wound more tightly, and hence, inhibit it from being transcribed. Or in other words, DNA methylation "turns off" genes.

non-coding RNA (ncRNA) contribute to the regulation of the chemical changes that affect chromatin structure.
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In humans, chromosomes come in pairs, and if they both code for the same genes, these are called homologues. Although they are the same traits (e.g. eye color) they may code for different versions (or alleles) of the traits (e.g. brown vs blue).

• Diploid - Any cell that contains a homologous pair of chromosomes. 
• Haploid - Any cell that does not contain homologues. 

Cellular Life Cycle: 

Most cells spend their life in the G0 phase. (Cells produce proteins during the G0 phase) 

Order: G0 phase --> G1 phase --> S phase --> G2 phase --> M phase --> back to G1 etc.

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Transcription: 

Eukaryotic transcription only takes place in the nucleus, and the mitochondria.

Includes three main stages:

• Initiation - Transcription factors (DNA binding proteins) identify a promoter on the DNA strand --> Transcription initiation complex --> RNA polymerase unzips the DNA double helix and creates a transcription bubble. 
• Elongation - RNA polymerase transcribes one strand of the DNA sequence into a complementary RNA nucleotide sequence. This transcribed strand is called the template strand or (-) antisense strand. The other strand is called the coding strand or the (+) sense strand, and it protects its partner against degradation. RNA polymerase moves from 3`-5` and builds new RNA strands from 5`-3`. 
• There is no proof reading mechanism for transcription processes, Moreover, errors in RNA are NOT called mutations, like they are in DNA errors. Errors in RNA are simply not transmitted to progeny. 
• Termination - Termination sequence is reached. It also involves special proteins. For exmple, the Rho proteins help to dissociate RNA polymerase from the DNA template.

Activators and Repressors - bind to DNA close to the promoter and either activate or repress the activity of RNA polymerase. This is a method of gene expression regulation. These proteins are often allosterically regulated by small molecules such as cAMP.
     -In eukaryotes, Enhancers are analogous to Activators, but require a much greater distance from the promoter.

Regulation of Transcription in prokaryotes:

(Prokaryotic) Several genes in a single transcript = polycistronic
(Eukaryotic) mRNA that includes only one gene per transcript = monocistronic

Operator + promoter + genes that contribute to a single prokaryotic mRNA = Operon
     -Example: lac operon in E.coli

E. coli prefers glucose as its energy source. The lac operon codes for enzymes that allow E.coli to metabolize lactose when glucose is not present in sufficient quantities. Activated when:

1. Glucose is scarce
2. Lactose is present

Low glucose --> high cAMP --> binds to and activates CAP (catabolite activator protein) --> binds to a CAP site located adjacent and upstream to the promoter --> activates promotor --> forms, transcribes and translates three proteins. 

     -A second regulatory site on the lac operon called the operator, is located adjacent and downstream to the promoter. If lactose is not present in the cell, a lac repressor protein binds to the operator, inhibiting the transcription of lac genes and preventing gene expression (gene repression). However, if lactose is available, it will bind to the actual repressor protein and won't allow it to bind to the operator. In other words, the presence of lactose induces the transcription of the lac genes only when glucose is not present. 

Note: 

Upstream = To the left (or towards the 5` end) 

Downstream = To the right (or towards the 3` end) 

Formal definition: An operon is a sequence of bacterial DNA containing an operator, a promoter, and related genes. The genes of an operon are transcribed on one mRNA. Genes outside the operon may code for activators and repressors (as seen in the picture above).
     -Promotor and gene for the lac repressor are located adjacent and upstream to the CAP binding site (as seen in the picture above).

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Modification of RNA:

-In eukaryotes, EACH type of RNA undergoes post-transcriptional processing. (mRNA post-transcriptional modification occurs in the nucleus)
-In prokaryotes, rRNA and tRNA go through post-transcriptional processing, but almost all mRNA is translated directly to protein, without going through post-transcriptional processing. (This is because the bacterial genome does not contain introns.)

Post-transcriptional processing of mRNA in Eukaryotes:

The primary nucleotide sequence is called the primary transcript (pre-mRNA or heterogenous nuclear RNA [hnRNA]).

• Even before the eukaryotic mRNA is completely transcribed, its 5` end is capped in a process using GTP. The 5` cap serves as an attachment site in protein synthesis during translation and and as a protection against degredation by enzymes that cleave nucleotides, called exonucleases. 
• The 3` end is protected from exonucleases by the addition of a long series of adenin nucleotides, called the poly A tail. After it's been added, it is said to be polyadenylated. 
• Before leaving the nucleus, portions of the primary transcript are excised through splicing. The transcript has introns and exons, with only the introns being the ones to be excised. (exons get expressed) This splicing process involves small nuclear ribonucleaoproteins, or snRNP's ("snurps"). This contains proteins and snRNA, where the snRNA acts as a ribozyme (an RNA molecule capable of catalyzing specific chemical reactions. One of the few enzymes that is not a protein.)
• This whole machinary complex formed from the snRNPs is called the spliceosome. 
• After excision, Alternative splicing allows the cell to incorporate different coding sequences into the mature mRNA. This creates a variety of mRNA molecules from a single DNA coding sequence. (This is the reason why there are more protein products than there are genes)
• The mechanism includes omitting certain exons, and including certain introns. Also includes a variable splicing site. (Only cut half of an exon etc.)
• Sequences that contain introns are associated with amplified protein production compared to sequences that lack introns.

*The separation of transcription-translation via the nuclear membrane only in eukaryotes allow an extra form of regulation. Since prokaryotes have no nuclear membranes, then transcription-translation happens simultaneously. 

All in all: Primary transcript --> Addition of 5` cap + 3` Poly A tail + Splicing + Alternative splicing --> mature mRNA.

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Translation:

The genetic code is degenerative, which means that more than one series of three nucleotides will code for one and and only one amino acid.
     -The reason we have a triplet code of three nucleotides is because a combination of two (4^2 = 16) won't make all 20 amino acids. However a a combination of three (4^2 = 64) does. This makes the code degenerative and unambiguous (since one codon always codes for one amino acid)

Start codon: AUG (codes for methionine)
Stop codons: UAG, UGA, UAA

-Nucleotides are written 5`-3`

Translation occurs in the cytosol, using a ribosome, which is either free-floating in the cytosol or attached to the outer surface of the ER to form rough ER. Composed of a small and large subunit which is made from rRNA and other proteins.

• Prokaryotic ribosomes are made from a 30S and 50S which has a combined sedimentary coefficient of 70S. These are smaller than eukaryotic ribosomes. 
• Eukaryotic ribosomes are made from a 40S and 60S which have a combined sedimentary coefficient of 80S. 
• Ribosomes are manufactured inside the nucleolus. (prokaryotes don't have a nucleolus)

tRNA renders the triplet code of the mRNA into a specific AA sequence. Each tRNA molecule has two distinct ends. One contains a series of three nucleotides, called an anticodon, which binds to the complementary codon sequence of the mRNA. The other end carries the AA that corresponds to that codon.
     -The first two base pairs in the codon and anticodon must be STRICTLY complementary. However, there is some flexibility in bonding at the third pair position. This is called wobble pairing, and helps explain why multiple codons can code for the same AA.

Includes the same three main stages:

• Initiation - With the help of initiation factors (co-factor proteins), the 5` end attaches to the small subunit of a ribosome. A tRNA containing the 5`-CAU-3` anticodon sequesters the AA methionine and settles into the P site (peptidyl site). This signals the large subunit to join and form the initiation complex. 
• Elongation - Ribosome slides down the mRNA strand one codon at a time in the 5`-3` direction, matching each codon with a complementary tRNA anticodon. This process requires the input of energy. Once a methionine-bearing tRNA has attached to the P site, a new tRNA, with its corresponding AA, attaches to the neighboring A site (aminoacyl site). 
• The C-terminal (carboxyl end) of methionine attached to the N-terminal (amine end) of the AA at the A site via a dehydration reaction. (2 molecules combine with the loss of 1 water) 
• The methionine subsequently moves to the E site. 
• This all happens until a stop codon reaches the P site. 
• Termination - Stop codon (nonsense codon) is reached. 
• When a nonsense codon reaches the A site, proteins known as release factors (other co-factor proteins) bind to the A site, allowing a water molecule to add to the end of the polypeptide chain. This allows the polypeptide to free from the tRNA and ribosome, and allows the ribosome to break up into its subunits to be reused later. 

As the polypeptide is being translated, it begins folding. This folding process is assisted by proteins called chaperones.

Post-translational modifications:

Affects which products ultimately become functional proteins. Sugars, lipids, or phosphate groups may be added to AAs to influence functionality. It can also be cleaved in one or more places.
     -Proteins translated by free-floating ribosomes function in the cytosol. Proteins synthesized by ribosomes in the rough ER are injected into the ER lumen, destined to become membrane bound proteins of the nuclear envelope, ER, Golgi, lysosomes, or plasma membrane, or to be secreted from the cell.
          -Translation begins on a free-floating ribosome. However, a signal peptide recognized by a protein-RNA signal-recognition particle (SRP) at the beginning of the translated polypeptide may direct the ribosome to attach to the ER, in which case it is injected into the ER lumen. Polypeptides injected into the lumen may then be secreted from the cell via the Golgi body or may remain partially attached to the membrane.

Genetic and Cellular Replication: Mitosis

Not all cells divide. Nuerons for example, primarily remain in the G0 phase.

-DNA replication takes place in the S phase.
     -This is a semiconservative process. (Each copy contains one strand from the original DNA, and one newly synthesized strand)
     -The mechanism is governed by a group of proteins called a replisome. It begins towards the middle of a chromosome at a site called the origin of replication. In prokaryotes, it usually takes place from a single origin on the circular chromosome. In eukaryotes, there are multiple origins of replication since the chromosomes are much larger. It is also a bidirectional process.

Replication Enzymes:

• DNA Helicase - Unwinds the double helix.
• DNA Polymerase - Synthesizes the new DNA strands. (Reads the parent strand in the 3`-5` direction, and creates strands in the 5`-3` direction.) 
• Primase - An RNA polyerase, creates an RNA primer approximately 10 ribonucleotides in length to initiate the strand. 
• Single strand binding proteins (SSBPs) - Prevents the single strand in the loop from folding back onto itself. 
• RNAase H - Removes RNA primer. 
• DNA ligase - Joins Okazaki fragments together. 

The continuous strand is called the leading strand.
The interrupted strand is called the lagging strand. (formed from a series of disconncted strands called Okazaki fragments)

Telomeres - are repeated nucleotide units that protect the ends of chromosomes. They can become shortened through repeated replication, which is a condition that has been linked to aging and disease.
     -Telomerase catalyzes the lengthening of telomeres in eukaryotic organisms. Prevents shortening.

One of the subunits in DNA polymerase is an exonuclease, which acts as a proofreader that makes repairs when it discovers any mismatched nucleotides.

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Mitosis:

Order: PMAT (Prophase, Metaphase, Anaphase, Telophase) Before any of this, there is Interphase.

• Prophase - Condensation of chromatin into chromosomes. Sister chromatids are joined together at their centers called centromeres (referring to location). Structures, called centrioles (where spindle microtubules come out of) located in the centrosomes move to opposite poles of the cell. Nucleuolus and defined nucleus disappear as the nuclear envelope breaks down. This is when the spindle apparatus begins to form, consisting of asters (microtubules radiating from the centrioles), and spindle microtubules connecting the two centrioles. The kinetochore is a structure of protein and DNA located at the centromere of the joined chromatids of each chromosome.
• Metaphase - Chromosomes align along the equator of the cell. This ensures that they will be separated such that each daughter cell receives one of each chromosome. 
• Anaphase - Sister chromatids split at their attaching centromeres and go to opposite sides of the cell. The shortening of the kinetochore microtubules pulls the sister chromatids apart. This split is termed disjunction. Cytokinesis, which is the ACTUAL separation of the cellular cytoplasm, may or may not commence toward the end of this phase. 
• Telophase - The nuclear membrane reforms, followed by the reformation of the nucleolus. Cytokinesis continues, resulting in two identical cells. 

Mutations:

Mutations in somatic cells are not passed down to offspring. Mutations in germ cells are.
     -Can be induced through agents called mutagens.

• Gene mutation - Alteration in DNA of a single gene. 
• Chromosomal mutation - When the structure of a chromosome is changed. 
• Somatic mutation - Mutation in a somatic cell (mutation of a single cell will have little effect)
• Germ cell mutation - Mutation in a germ cell (mutation in germ cells can have large consequences for the offspring produced.) 
• Point mutation - Mutation that changes a single nucleotide in a double strand of DNA. 
• Base substitution mutation - When one nucleotide is swapped for another during DNA replication. (purine with purine, or pyrmidine with pyrimidine = transition mutation, while purine with pyrimidine = transversion mutation) 
• Addition mutation - Inserting a new nucleotide into the sequence. 
• Deletion mutation - Deleting a nucleotide from the sequence. 
• Additions and deletions have profound effects on the function of the affected gene. 
• Neutral mutation - If a mutation has no effect on an organisms fitness or function of a protein.
• For example, changing the codon AAA to AAG through a base substitution would still result in the amino acid lysine.
• Silent mutation - Neutral mutation in which the AA sequence is unchanged. 
• Missense mutation - Occurs when a base substitution changes a codon. 
• Can be neutral and result in completely functional proteins. This results in the translation of a different amino acid.
• Nonsense mutation - Occurs when a change to the nucleotide sequence creates a stop codon where none previously existed. 
• Tends to have very serious consequences because they usually result in truncated, non-functional proteins. 
• Frameshift mutation - Occuts when the deletions or additions occur in multiples other than three. 
• This changes the reading frame of the code. Most often results in completely non-functional proteins as well. 

Mutations at the level of the chromosome:

Mutations at the chromosome level almost always cause serious consequences for the organism. 

• Deletions - When a piece of the chromosome breaks off, or when a piece of lost during homologous recombination and/or crossing over events. 
• Duplication - Occurs when a DNA fragment breaks free of one chromosome and incorporates into a homologous chromosome. 
• Gene duplication (or gene amplification) can increase the amount of a gene's product. 

-Gene deletion or duplication can occur with entire chromosomes (aneuploidy) or even entire sets of chromosomes (polyploidy). 

• Translocation (reciprocal) - When a segment of DNA from one chromosome is exchanged for a segment of DNA on another chromosome. 
• In inversion, the orientation of a section of DNA is reversed on a chromosome. Translocation and inversion can be caused by transposition, which takes place in both prokaryotic and eukaryotic cells. Dna segments called transposable elements or transposons can excise themselves from a chromosome and reinsert themselves at another location. (These can contain one or several genes) 
• When moving, the transposon may excise itself from the chromosome and move; copy itself and move; or copy itself and stay, moving the copy. (Transposition is a mechanism by which a somatic cell can alter its genetic makeup without meiosis.)

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DNA repair can be achieved by: 

• Subunit of DNA polymerase - Correcting mismatches in DNA sequences.
• Direct repair - In which damaged nucleotides are chemically changed back to their original structures.
• Excision repair - In which damaged nucleotides are removed and replaced.  

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Cancer:

Certain genes that stimulate normal growth in human cells are called proto-oncogenes. These can be converted to oncogenes, by mutagens (carcinogens) such as UV radiation or chemicals, or through random mutation. The genome also contains tumor supressor genes that help regulate normal cell growth.

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Meiosis:

Takes place solely in the reproductive organs.

• Gametes - haploid reproductive cells

-Only the spermatogonium and the oogonium undergo meiosis in humans. 

• Meiosis I - Separates homologous chromosomes to produce two haploid cells. (Reduction division)
• Prophase I - Homologous chromosomes line up alongside each other, matching their genes exactly. They exchange sequences of DNA via crossing over. The genetic recombination that occurs in eukaryotes during crossing over produces genetic variation. The side by side homologues exhibit a total of four chromatids, and are called tetrads. The 'X' shape created where the two chromosomes are attached is called a chiasma. Genes closer together are more likely to cross over than genes farther apart from each other. This is called gene linkage. There are single and double cross overs. (Gene mapping helps determine the locations and relative distances of genes on chromosomes) 
• Single crossover - Chromosomes exchanging sections of genetic info just once. 
• Double crossover - Chromosomes trade a segment once and then trade back a sub-section of that segment so that each chromosome regains some of its own original genetic info. 
• Metaphase I - The two homologues remain attached, and move to the metaphase plate. 
• While in mitoses each chromosome lines up along the plate single file, in meiosis, the tetrads align on the metaphase plate. 
• Anaphase I - The homologous chromosomes each separate from their partner, independently assorting to create two haploid cells. 
• This is in  contrast to anaphase in mitoses, where identical sister chromatids separate. 
• Telophase I - A nuclear membrane may or may not reform, and cytokinesis may or may not occur. 
• In humans the nuclear membrane does not reform, but cytokinesis does occur. 
• Meiosis II - Much like mitosis
• Prophase II - Much like mitosis
• Metaphase II - Much like mitosis
• Anaphase II - Much like mitosis
• Telophase II - Much like mitosis 

if the chromosomes do not split in anaphase I or II, this is called nondisjunction. (Remember that the splitting of the chromosomes during anaphase is called disjunction) 

     -A primary nondisjunction (nondisjunction in anaphase I) will result in one of the cells having two extra chromatids, or in other words, a completely extra chromosome. The other will be missing. 

     -Nondisjunction in anaphase II will result in one cell having one extra chromatid and one cell lacking one chromatid. 

     -Down syndrome is caused by nondisjunction of chromosome 21. 

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Meiosis is not the only process by which nuclear genetic information is passed. Extranuclear inheritance, which is also called cytoplasmic inheritance (e.g. in the mitochondria and chloroplasts), takes place via its own mechanisms. Genetic leakage, the flow of genetic information from one species to another, can also take place under certain conditions. 

Other definitions: 

• F1 generation - First generation to be tested. 
• Dominant gene - Gene that trumps recessive genes. 
• Recessive gene - Gene that is trumped by dominant genes. 
• Locus - Position of a gene. 
• Allele - Type of a particular trait. 
• Wild type - Most common allele for a certain trait. 
• Genotype - Genetic makeup of a species. 
• Phenotype - Expression of a trait. 
• Complete dominance - Where dominant genes mask recessive genes in the phenotype. 
• Homozygous - Either 'PP' or 'pp' 
• Heterozygous - 'Pp', also called a hybrid. 
• Law of Segregation - First law of heredity, which states that alleles segregate independently of each other when forming gametes during meiosis. 
• Also, the phenotypic expression of the alleles is not a blend of the two, but an expression of the dominant allele. (The principle of complete dominance)
• Penetrance - Refers to the probability of a gene or allele being expressed if it is present. 
• In complete dominance, the penetrance of the dominant allele is 100%. 
• Expressivity - Measure of how much the genotype is expressed as a phenotype. 
• Incomplete dominance - When a heterozygous individual exhibits a phenotype that is intermediate between its homozygous counterparts. (For example, a cross between red flowers and white flowers producing pink flowers)
• Co-dominance - When both traits are expressed. (White cat and black cat mate to make white and black cat. Blood types work this way too.) 
• Law of Independent Assortment - The second law of heredity, which states that genes located on different chromosomes assort independently of each other. 
• Barr body - Inactive X chromosome in a female somatic cell. 
• Gene pool - Total collection of ALL alleles in a population.
• Hardy-Weinberg equilibrium - Assumption that there is no net change happening in allelic frequencies over time. Conditions include: (In other words, for evolution not to happen)
• No Selection for the fittest organism
• Random mating
• Large population
• Immigration or emigration must not change the gene pool
• Mutational equilibrium 

p^2 + 2pq + q^2 = 1 predicts the genotype frequencies of a gene with only two alleles in a population in Hardy-Weinberg equilibrium.

Because there are only two alleles, then p + q = 1 

-Phenotypic ratio of a dihybrid cross: 9:3:3:1
-Genes located on the sex chromosome are said to be sex-linked.