Genes, Genomes, and transposable elements

L11

genome

the entirety of an organism’s hereditary info

• usually DNA

what is the eukaryotic genome composed of?

• coding DNA (islands)

and

• non-coding DNA (open ocean)

does genome size correlate to genome complexity? what account for diffce in genome size?

not necessarily… the difference in genome size is largely due to the amount of non-coding DNA

what leads to gene density variability among eukaryotes?

the size of intragenic (i.e. introns) and intergenic regions (i.e. between genes)

how much of the human genome do coding sequences account for ?

abt 3%

gene

• the entire nucleic acid sequence that is necessary for the synthesis of a functional product (polypeptide or RNA)

• genes are transcribed

what do exons of a gene contain

• the coding region or Open Reading Frame (ORF)

  • contains codons telling ribosome to start/stop translation — aka protein synthesis

control regions

promoter and cis- regulatory factors

• are found either upstream or downstream to coding regions

• genes need these to control regions for transcription to occur 

introns

separe the exons and are spliced out during mRNA processing

• aren’t used for the final product

transcription unit

a region in DNA bounded by an initiation (start) site and termination site that is transcribed into a single primary transcript

• aka the sequence that is actually copied in RNA

introns can vary in size. what can we say about them in human genes?

• many human genes >10kB in length

  • some are over 1Mbp

• vast majority (>95%) of an average human gene is non-coding

  • mostly intron sequences

    • represent about 42% of the totla length in the genome total

what is the function on introns

• eukaryotic genes are alternatively transcribed and processed

  • generates multiple difft transcripts from the same gene

isoforms

the multiple forms of a protein produced by alternative splicing

solitary or single copy genes

• 25-50% of protein-coding genes

• represented once in the genome 

gene family

• made up from a set of related genes formed by duplication of

  an original single-copy gene

• called duplicates, occur in multiple copies

what can we say about the sequences of protiens with similar functions

• they often contain similar AA sequences that encode functional domains

  • unlikely that this would have been generated independently => they must come from the same gene

what is an example of a sequence alligment technique we can use to find nucleic acid and protein sequence similarity 

BLAST

what is the difference among genome size in species due to ?

• mostly due to difft amts of non-coding DNA and Transposable elements

• complexity cannot be explained by number of genes

gene duplication

• an imp process in evolution

• new gene copies either

  • evolve a new function

  • degenerate over time and lose their function => become pseudogenes

what does the comparison of related protein seqs in difft spp illuminate?

• the evo relationships between these

orthologs

the same protein in different species (α-tubulin in humans and flies)

Paralogs

closely related proteins in the same species (α-tubulin and β-tubulin

in humans)

intragenic non-coding DNA

includes

• introns

  • are spliced out

• UTRS

  • are part of mature mRNA but non translated

Simple Sequence Repeats SSrs

• 6% of the genome

• first group part of non-coding DNA

• includes

  • minisatellite DNA

  • microsatellite DNA

minisatelite DNA (an SSR)

• Repeat units are ≈14 to 100 bp in length

• 20-50 tandem repeat units

• Arrays of 1 to 5 kbp in length

• Often in centromeres and telomeres

microsatellite DNA (an SSR)

• Repeat units are typically 1 to 4 bp in length

• Arrays of up to ≈600 bp in length and composed of tandem repeat units

• Sometimes found in transcription units

• Expansion underlie several neuromuscular diseases

  • like myotonic dystrophy

  • and spinocerebellar ataxia

how does slippage occur in DNA?

during the replication of long repeats

• polymerase stalls

slips or looses its place on the template strand

• causes the template and synthesized strand to missallign leading to temporary loop formation

• synthesis resumes

  • the restabilization can result in the addition of more repeats than were in the OG template — this is called repeat extension

how is gene slippage associated to neuromuscular diseases?

• e.g. Huntington’s

• results in the productuion of protein that form toxic aggregates in neuronal cells

how can the hypervariable nature of SSRs be exploited for DNA fingerprinting protocols? (for paternity determination or criminal identification)

• SSRs can be amplified by PCR ( or studied by southern blot)

• the n° of repeats is determined by high res gel electrophoresis 

what are the 2 major classes of Transposable Elements (TEs) we studied?

• DNA Transposons

  • 3% of genome

  • just jump from one position to another

• Retrotransposons

  • 40% of genome

  • copy themselves threough an RNA intermediate before moving around (that’s why there are more)

Transposable (Mobile) DNA Elements aka Jumping Genes

• move within the genomes by difft mechanisms

• influenced evolution

• can cause mutations leading to disease

• originally identified by Barabara McClintock by studying coor pattern formation in maize (cause by production of the pigment anthocyanin)

what is the mechanism of transposition 

• DNA transposases inserted through cut-and-paste mechanism

(1)

• Transposase makes blunt ended cuts in donor DNA

• and staggered cuts in taget DNA 

(2)

• transposase ligates IS10 to 5’ single stranded ends of target DNA

(3)

• cellular DNA polymerase extends 3’ cut ends and ligase joins extended 3’ ends to IS10 5’ ends

• ligating the blunt-end transposon sequence in the staggered-ended recipient seaquence reslts in a short duplication of DNA (9bp)

Transposase

enzyme that catalyzes the insertion of DNA transposons

how can a DNA transposon increase its copy number (despite its cut-and-paste mechanism)

• during S phase

• if transposon moves from region that has replicated to one that has not then the copy number will increase by one in one of the daughter chromosomes (one daughter molecule will have two copies fo the tranposon)

LTR (Long Terminal Repeats) - a type of retrotransposon

• inserted at 440,000 sites, abt 8% of our genomes

• are similar to retroviruses but lack envelope proteins

• their protein coding region encodes

  • reverse transcriptase

  • integrase (similar function to transposase of DNA transposons)

  • and other proteins

mechanism od copy-and-paste of LTRs

• LTRs are first transcribed

  • generating an RNA copy of most of their sequence, excluding part of the ends

• LTRs require a retrotranscriptase to convert the RNA molecule into DNA through a multiple step process.

  • This process occur in the cytoplasm.

• A molecule of tRNA is used as a primer in the process

  • RNA is complementary to tRNA which is why it can be used as a primer

• DNA is then imported to the nucleus in complex with integrase (a protein related to transposases used by DNA transposons)

  • Ingrase will mediate the insertion into the genome using a similar mechanism as transposases do for DNA transposons

what are two types of nonviral retrotransposons

• LINEs

• SINEs

LINEs

• 900,000 of these in our genome - represents abt 21% of it

  • these are the most common retrotransposons

• contain two open reading frames

  • ORF1

    • for RNA binding

  • ORF2

    • encodes a reverse transcriptase and a nuclease, mediating the insertion

SINEs

• abt 300bp in length (much shorter than LINEs)

• occur at 1.6million sites in our genome -  accounting for abt 13% of it

• do not contain ORFs

• insert themselves in the genome similarly to LINEs w AT rich regions but are parasites of LINEs because use those regions to insert themselves into the region but do not code proteins

Alu

• the most common SINE — aka most common repeated sequence in the genome

• probably evolved from a non-coding RNA gene

how does the DNA insertion of LINEs work?

• RNA is produced and exported from the nucleus

• ORF1 (and RNA-bonding protein) and ORF2 (Reverse transcriptase and nuclease) are translated and bind LINEs RNA

• RNA-protein complex imported to the nucleus

• Nuclease cuts DNA at an AT-rich sequence and uses the DNA ends as primers

• No transposase or integrase used

how do SINEs insert themselves?

• they use ORF1 and ORF2

how do TE mvmnts lead to genome changes?

• Recombination between repeated elements can shuffle exons and produce new genes with new combination of existing exons

• helpful because can do recombination with non-homologous chromosomes

What can transposons and LINEs carry with them when they move that causes shuffling?

they can carry unrelated flaking sequences