PCB4522 Module 1 Meet the Molecules Pt 2

Genome

Complete set of chromosomal DNA in a nucleus, cell, or organelle.

Prokaryotes: main chromosomes, not plasmids;

Eukaryotes: nuclear genome ( linear chromosomes), mitochondrial genome, and chloroplast genome (plants).

The -omes

1. Transcriptome: full set of RNAs;

2. Proteome: full set of proteins;

3. Metabolome: full set of metabolites and other small molecules;

4. Interactome: full set of protein-protein interactions present;

5. Microbiome: full set of microbes in/on an organism or tissue.

REVIEW THESE TERMS!

Karyotype, allele, haplotype, locus (p. loci), epigenome, linkage.

Genomics: study of genomes

1. sequencing and assembly;

2. content, structure, and interactions;

3. comparisons within and between populations and species;

4. evolution.

Genome Content and Characteristics

1. genome size;

2. chromosome number/ karyotype;

3. gene number;

4. repetitive DNA

See lec 2 slides p7-p14 for genome size, chromosome number, and gene number diversity among species.

Repetitive DNA

1. Tandem repeats: satellites, minisatellites, and microsatellites;

2. Dispersed repeats: retrotransposons and DNA transposons.

Tandem repeats

1. microsatellites: less than 10 bp motifs;

2. minisatellites: 10-100 bp motifs;

3. Satellite DNA at telomeres and centromeres: 100s-1000s bp motifs;

4. rDNAs;

5. CRISPRs for prokaryotes.

Dispersed repeats

1. Retrotransposons: class I, copy and paste;

2. DNA transposons: class II, cut and paste.

Physical interactions within a genome

1. chromosome territories;

2. linkage versus physical distance;

3. topologically associating domains (TADs) (euk only).

TAD formation and significance

Certain regions within chromosomes are highly associated with each other (cell-type specific);

Distribution measured by Chromosome Conformation Capture (3C);

Cohesin + CTCF -> drives TAD architecture by repressing or promoting enhancer-promoter interactions -> dictates TAD boundary.

End of Lec 3

STUDY STUDY STUDY

Genome evolution

1. Codon bias;

2. Gene identity;

3. Collinearity, synteny, and linkage groups;

4. Somatic genome vs germ line genomes;

5. Genome duplications;

6. Endosymbiosis and horizontal gene transfer;

7. Genetic drift

Codon Bias

Uneven usage of different codons to make the same amino acid;

Causes: mutational bias, tRNA abundance, and RNA stability.

Gene Origin/Identity

They could be:

1. full duplicates of other genes;

2. partial duplicates of other genes;

3. fusions of genes;

4. frameshifted genes;

5. duplicated or translocated promoter (= a "new" gene).

Gene homology

i.e., shared, derived ancestry.

We study the DNA and amino acid sequence alignment and synteny to infer gene relationships.

The conservation of gene order and content are assessed between genomes of different species to identify homologous genes or regions of a chromosome.

1. Orthologs: homologous genes in 2 different genomes;

2. Paralogs: previously duplicated homologous genes in the same genome;

3. Homologs: general term for homologous genes.

Collinearity

Homologous genes are in the same order on a chromosome in two different genomes.

Synteny

Homologous genes are co-localized on chromosomes in two different genomes.

Linkage Groups

Groups of genes that exhibit synteny between genomes.

Gene and Genome Duplications

Since oftentimes duplicated genes become nonfunctional, those that persist are thought to provide more "raw material" for evolution.

Somatic cells

All cells except gonads;

Mutations in the somatic cell genome are not heritable;

Germline cells

The cell lineage that leads to gametes;

Mutations are heritable.

Endosymbiosis

When a cell engulfs another cell and does not consume it, instead it forms a partnership through many generations e.g. mitochondria used to be an aerobic, heterotrophic bacteria.

Organelle Genomes

Almost all Eukaryotes have at least one organelle with its own genome->plastid;

The vast majority of eukaryotes have mitochondria;

Have prokaryotic characteristics:

Circular, operons, bacterial rDNA, no Fis/HU in mito (chloroplasts have HU-like protein), instead use transcription factor TFAM.

Horizontal Gene Transfer from plastids

Genes critical to the organelle's function often migrate from the plastid genome to the host's nuclear genome.

Horizontal Gene Transfer, Cont'd

1. conjugation: sex pilus;

2. transduction: DNA hitchhike w/ viruses;

3. transformation: uptake from environment.

HGT is less common in eukaryotes; see slides p41-p44.

Genetic Drift

The change in frequency of an existing genetic variant in a population due to chance.

Genetic Drift Cont'd

Small population, lower reproductive rate, founder effect, and bottleneck --> strong genetic drift.

End of Lec 4

Only one more lecture to go!

Common methods in molecular biology listed

PCR, Gel electrophoresis, Precipitation, Cloning and DNA sequencing engineering, Sanger sequencing, In situ hybridization, Morpholinos and RNAi, Mutagenesis, and Transgenesis.

PCR, Polymerase Chain Reaction

Make copies of a specific interval of DNA.

Cocktail ingredients: template DNA, primers, Taq pol, buffer, dNTPs;

Temp: denaturation = 94C, primer annealing = 54-60C, elongation = 72C.

Gel electrophoresis (nucleic acid)

Nucleic acid samples are loaded into a ~1-3% agarose gel, and electricity is applied. Molecules migrate based on mobility, which is determined by size/conformation/etc.

Gel electrophoresis (protein)

Protein samples are loaded into an acrylamide (PAGE) or agarose gel, and electricity is applied; The mass/charge ratio, shape/conformation, polarity, and binding interactions determine mobility.

Precipitation

Used to clean and isolate nucleic acids or proteins;

Add chemicals that force your molecule of interest to make it insoluble, spin it down into a pellet, then remove the solution.

Cloning and DNA sequencing engineering

1. Restriction enzyme cloning;

2. Modern methods: Gateway cloning and Gibson assembly.

Sanger sequencing

Determines the sequence of 500-1000 nucleotides downstream of a priming site; uses ddNTP to end elongation; fast and cheap.

Sanger sequencing strategy

ddNTPs (di-deoxy, missing 2' and 3' OH) act as chain terminators. We infer the identity of the base in the terminated strands to infer the sequence of bases.

In situ hybridization

Allows for spatial visualization of RNA transcripts in an embryo or tissue;

ISH genomic: Allows for detection of specific sequences in chromosomes.

Morpholinos and RNAi

Short, synthetic nucleic acids are administered to an organism and interfere with normal translation or transcript splicing.

1. Morpholinos: single-stranded "nucleic acids" that are relatively stable and inert,~25 nt long, block mRNA splicing or translation;

2. RNA interference: short interfering RNAs (siRNAs): double-stranded RNAs that mimic miRNA pathway, ~21 nt long.

Mutagenesis

1. targeted mutagenesis: reverse genetic screen

2. untargeted mutagenesis: forward genetic screen

Transgenesis

Integrating a reporter transgene or an experimental transgene can integrate foreign DNA into an organism.

1. targeted techniques: homologous recombination, Cre-Lox

2. untargeted techniques: Tol2, I-SceI.

Transgenics are often mosaic in the F0 generation.

End of Lec 5

Good luck with your exam!