Molecular Biology (BIOB11) Midterm Review

Mitosis

Mitosis

Occurs in somatic cells.

You end up with diploid cells that are genetically identical.

Meiosis

Occurs in two steps.

The 1st has two chromosomes (homologous chromosomes) that are pulled apart

The 2nd looks like mitosis with sister chromatids being pulled apart into two different cells.

End up with 4 haploid cells that are genetically non-identical

Laws of inheritance

Individuals have two copies of each genes called gametes (a single copy of every chromosome). Dominant alleles, homozygous, heterozygous.

Dominant alleles

Determine phenotype

Homozygous alleles

AA or aa

Heterozygous alleles

Aa or aA

Law of segregration

An individual’s maternal and paternal chromosomes move apart from one another during gamete formation. One gamete carries one allele for each gene.

Law of independent assortment

Segregation of a pair of alleles for one trait (gene) has no effect on the segregation of alleles of another trait.

This occurs in meiosis 1. Creating genetic organisms, increasing fitness. Possible due to crossing over

Homologous chromosomes

Tetrad or bivalent pair in meiosis. Pair together in meiosis 1 and are pulled apart.

Crossing over

Breaking and rejoining of pieces of DNA. Chromosomes form a chiasm and this process occurs mostly in homologous chromosomes, although it could potentially happen in other stages like heterozygotes.

Genes located close to each other MAY be linked.

Kinetochores

Microtubules. Attach at centromere and haploid daughter cells post crossing over have a more diverse genetic makeup.

Trisomy

Issues in meiosis/mitosis with too many chromosomes. They couldn’t separate.

Issues in meiosis/mitosis

Trisomy, unreciprocated crossing over (too much/little regulatory DNA), two centromeres (cancer), extra mitotic spindle does third division.

DNA

Carrier of heritable information. Needed for viruses to transfer. Separate to find negative strain. Franklin found out helical structure.

Right hand spiral with minor and major grooves.

A/T

2 hydrogen bonds. Lower melting point

G/C

3 hydrogen bonds. Higher melting point.

A/G

Purines

T/C

Pyrimidines

Eukaryotes

• Multiple origins of DNA

• Use telomerase to maintain chromosome ends

• Nucleosomes

• DNA in nucleus

  • DNA in mitochondria, pathogens and plastids

Prokaryotes

• Have very little non-essential DNA

• Archae and bacteria

• Circular chromosome, nucleoid region.

• Horizontal gene transfer

Eukaryotes and Prokaryotes

• Use DNA as a carrier of heritable DNA

• Lagging strand DNA made with Okazaki fragments

• DNA → RNA → Proteins

• Metabolism/take in energy

• Cell division

Genomes

Genes that perform the same functions.

Size is not correlated to amount of genes.

Gene density

Eukaryotes < mitochondria < prokaryotes

Composition of human genes

LINEs, SINEs, introns, exons, unique sequences, transposons, repeated sequences.

LINEs

Long interspersed nuclear elements.

Repeated sequences/transposons.

SINEs

Short interspersed nuclear elements.

Repeated sequences/transposons.

Transposons

Mobile genetic elements that move around and jump around the genome

Repeated sequences

LINEs, SINEs (transposons), simple sequence repeats, segmental duplications

Nucleus

6.4 billion base pairings in 46 chromosomes. 2 meters of base pairings. 1 base pairing per 6 H2O molecules. All stored in here.

DNA supercoiling

After becoming a double helix, DNA twists on itself even more. Becomes very tense and opening causes tension. Increased stability, compact, allows for unwinding of sections, relieves stress.

Positive: overwound

Negative: underwound

Nucleosomes

Eukaryotic DNA is associated with histones to form chromatin.

Made of 8 histones (octomer) and 2 wraps of DNA. 200 base pairs per histone.

Histone

Highly conserved proteins, rich in amino acids. High positive charge. start with a dimer and form an octamer.

Histone fold/histone handshake

Different sequences in the histone tail where modifications occur, regulatory modification. This tells the cell how to organize DNA.

H1

Linker histone. Links DNA together. Amount of H1 in there affects how tightly histones package together.

30 nm fold

Made up of the H1 (linker histone) and core nucleosome

Looped domain

Occurs following chromatin. Forming scaffolds of 30 nm fibres in loops to position chromatin into 700 nm folds, then 1400 nm fold, the 10 000 nm fold.

Euchromatin

Less condensed, toward centre, accessible for protein binding and transcription. All due to sections becoming less compact at interphase following mitosis.

Heterochromatin

More condensed, towards periphery, not much functional activity. At least 10%. The centromere + midsection and the telomeres.

Constitutive heterochromatin

Permanently silent, like the telomere and centromere. Repetitive DNA. Compact all the time.

Facultative heterochromatin

Active only during certain areas of an organism’s life. Happens as cell and body develop and while cell differentiation occurs. Inactivated during certain phases of an organism’s life.

X-inactivation.

Epigenetic inheritance

Occurs on top of the genome with no change to nucleotide DNA. Vital for developing tissues. Helps guide formation of heterochromatin and euchromatin. Histone coding.

Histone coding

Modifications of histone tails can disrupt or stabilize nucleosome assemblages. This is known to regulate chromatin structure and how condensed a region is

Acetylation

Leads to more open structure. Becomes uncondensed when H2, H3, and H4 have this.

Methylation

Leads to less transcription. Becomes more condensed when H3 and H4 have this.

Stabilizes nucleosomes and can prevent proteins from binding to DNA sequences and prevent transcription.

Histone acetyltransferases (HATs)

Acetylate histone proteins by transferring acetyl group from actetyl-CoA to specific lysine residues. Associated with euchromatin.

Histone deacetylases (HDACs)

Removes acetyl group

Histone methyltransferases (HMTs)

Add methyl groups to lysine or arginine residue. Associated with heterochromatin.

Histone demethylases

Removes methyl groups

Epigenetic memory

DNA methyltransferase can add the methyl group to DNA at sites where C is followed by G (5’ to 3’ CpG)

DNA is methylated as it is replicated so that methylation can be passed to daughter cells.

Silencing genes by heterochromatin occurs in regions and is maintained in replicated DNA (position effect).

Post-DNA replication

Epigenetic signals that regulate chromatin are propagated through space and time (as they are changing while they grow, they need to be maintained).

Reader complex

“reads” histone code and positions and activates “writer” enzymes that can act on adjacent DNA/histone.

On the histone code they work with Chromatin remodelling enzymes.

Chromatin remodelling enzymes

Works with reader/writer complexes. Perform functions like altering position of nucleosome in DNA, removing histones, switching in histone variants associated with particular functions.

Barrier protein

Certain DNA sequences recruit protein complexes that block spread of reader-writer complexes and separate chromatin into different domains. Create physical barriers. Different transcription activation regulation.

Protein coding sequences

More highly conserved than genome size and organization.

Error rate

Bacteria’s is 3 mistakes for 10^10 nucleotides copied

Human’s is 1 mistake for 10^10 nucleotides copied

Point mutation

Switching one nucleotide for another nucleotide

Large-scale rearrangements

Include deletions, duplications, inversions, and translocations.

Tandem repeats

To be highly repetitive you would need to have 10^5 copies of sequences repeated over and over without interruption

Satellite DNA

5-500 base pairs in tandem

Minisatellite DNA

10-100 base pairs with up to 3000 repeats. Highly variable. DNA fingerprinting. Criminal and paternity tests.

Microsatellite DNA

1-5 base pairs in clusters, 10-40 base pairs scattered evenly. Highly variable mutation. Closely related populations. Good for phylogenetic trees.

This type of instability contributes to progression of diseases.

Moderately repetitive DNA

About 20-80% of genome depending on the organism. Repeats a few times to tens of thousands of times. Can include genes or non-coding DNA. rRNA and histone.

Repetitive DNA sequences

Unstable. Easily expanding and shrinking due to slippage, where you hydrogen bond with the wrong base pair. Misalignement occurs and leads to deletions or expansions.

Regions of synteny

A block that carry genes in a conserved order

Intrachromosomal rearrangements

Usually in euchromatin, predisposed to large deletions, inversions or further duplications. Occur within a chromosome.

Interchromosomal rearrangement

Common in pericentromeric or subtelomeric areas. Between chromosomes.

Jumping Genes

DNA moves itself, transposons and mobile DNA.

Mobile DNA

DNA that moves from one place to another in the genome. This genetic rearrangement is called transposition and mobile genetic transposable elements. Transposition contributes to repetitive DNA sequence.

DNA Transposons

Cut and Paste.

Catalyzed by transposase enzyme. Creates repetition. Inverted repeats on the end of the transposon are required for recognition by transposase and excision from the donor DNA. The direct repeat is generated in the recipient DNA.

Retrotransposons

Copy and Paste. Involves RNA intermediate. LINEs and SINEs. Can encode reverse transcriptase enzyme to catalyze production of DNA and RNA.

RNA → cDNA → DNA

Exon shuffling

Transposons can pick up other sequences. If your genes pick up the transposon, it is more likely to land in an intron. Transcribed and then reverse transcribed, and it could also transcribe additional sequences.

Unequal crossing over

In meiosis, the genes misalign and one gene gets too much gene and does not reciprocate the DNA cross. Successful one leads to tandem arrays in DNA.

Gene Duplication

Point mutation changes one and that creates a new allele, may function just slightly differently. Creates redundancy. Having two genes and mutating one won’t affect the organism, as there is an original copy.

Horizontal gene transfer

Creates genes. Small portions get added in. Very damaging (no offspring, or offsprings die).

Orthologs

Speciation given two separate species. Different species evolved from a common ancestral gene by a speciation. Lineage splitting.

Paralogs

Gene duplication and divergence. Gene copies created by a duplication event within the same genome

Homologs

Any genes that are similar due to common ancestry (orthologs and paralogs)

Globin genes

Lots of mutations. Similarities in different functions. Lots of DNA similarities, but also some divergences. In humans they are orthologs and homologs.

Pseudogenes

When we have redundant replication and mutations accumulating in genes to the point that they’re now non functional.

Single Nucleotide Polymorphisms (SNPs)

Most common mutations. In protein coding regions, they contribute to phenotype differences (alleles). Changes on nucleotide and its base pairings.

Always inherited in a group thus we can track them. Help understand genetics.

Understand why things run in family.

Haplotypes

A particular combination of alleles on a chromosome that are inherited together.

Copy number polymorphisms (CNPs)

Differences in the number of copies of a particular sequence. In protein coding region, extra copies means more protein, and there is a phenotypic difference.

Structural variation

Large segments of DNA change by duplication, inversion, deletion, etc.

DNA polymerase

Adds new nucleotides to a growing DNA strand in 5’ to 3’ direction. Add new nucleotide onto a correctly positioned 3’OH. Has a catalytic site for editing (proofreading function), and so a mutation is detected and DNA is pushed into editing site.

Strand-directed mismatch repair

Recognition of a mismatch, identification of newly synthesized strand, removal of incorrect nucleotides from new strand, resynthesis of excised section, ligation to seal DNA backbone. MutS recruits.

Renaturation

Critical for RNA transcription and DNA replication. Re-association of hydrogen bonds.

Denaturation

Heats up to get enough heat energy to break and pull bonds apart. There is a small temperature range. Called DNA melting. Monitor UV light absorption. We get no change for awhile, and then after a specific temperature, then DNA absorbs a lot of UV light.

DNA renaturing graph

X-axis: Initial DNA concentration (C0) by time of reaction (t)

Y-axis: fraction of original DNA concentration that is renatured (dsDNA)

Replication fork

Points where a pair of replicating segments come together and join. 2 replication forks for every origin of replication.

DNA replication system

Helicases, single stranded DNA-binding proteins (SSBs), topoisomerase, primase, DNA polymerase(s), and DNA ligase.

Helicase

Opens up the DNA double helix ahead of the replication fork, making the template strand accessible. A-T rich sequences and their hydrogen bonds react with this.

Single-stranded binding proteins (SSBs)

Single stranded DNA has a tendency to fold on itself to make hairpins, and the this can help to stop the DNA from refolding. They bind and stabilize single stranded DNA after the helicase unwinds the double helix.

DNA Primase

DNA polymerase can’t start from nothing, and it needs 3’OH to build from. This starts a short segment of RNA to then build off of, they will be removed, but they allow DNA polymerase to start (about 10 nucleotides).

Topoisomerase

When double helix is opened up there is overwinding ahead of the fork and this aids it. Replication requires unwinding of DNA which causes torsional stress (positive supercoiling), which can stall replication. This relieves the stress and can be ahead of helix.

Topoisomerase 1

Catalyzes breaking/nicking of one DNA strand to allow for rotation (relieve strain)

Topoisomerase 2

Catalyzes as a double strand break, detangling DNA.

Sliding clamp

DNA polymerase has a tendency to fall off the DNA template and this prevents that. It is associated with polymerase to keep it in association with the DNA molecule. Needs clamp loader to open up ring and add it to DNA.

Okazaki fragments

DNA polymerase CAN’T synthesize 3’ to 5’ and these aid in that. Synthesis of each fragment waits for parental strand to separate and expose additional templates (lags behind leading strand synthesis). Primase adds RNA primer to each segment.

DNA polymerase 1

both 5’-3’ and 3’-5’ exonuclease can degrade DNA or RNA

Replisome

We have to keep everything coordinated and going at the same time. A multiprotein complex with contact to both leading and lagging strand to keep their replication coordinated.