GENOMICS 2.2: Structural and functional elements of the genome

What is the size range of prokaryote genomes?

Relatively small: 0,2-13 Mbp

What is the size range of eukaryote genomes?

Big: 3 Mbp - 700 Gbp

What is the genome organization and ploidy of prokaryotes?

• Circular genomes with accompanying circular plasmids

• Haploids (singular copy of the genome)

What is the genome organization and ploidy of eukaryotes?

• Nuclear and oragnelles enclosed with membranes

• Nuclear genomes: linear, organized in several chromosomes

• Organelle genomes: circular, reminiscent of bacterial

• Haploid (n) (gametos), diploid (2n), or polyploid (normally in plants)

What is the C Value?

The C value is the genome size of the haploid genome (in pg or Gb) → it allows us to compare genomes from different species

What is the C Value paradox?

The range of C values does not correlate with the complexity of organisms → we would expect that the more complex the genome the bigger it would be, but that is not always the case

Which organism has the biggest genome? And the second biggest?

1. Tmesipteris oblanceolata → fork plant (160 Gbp)

2. Paris japonica → canopy plant

How are eukaryotic nuclear genomes organized?

• Organized in chromosomes

• Each chromosome is a linear DNA molecule

• Number of chromosomes varies across species → similar to the C-value paradox, the number of chromosomes does not equal to complexity

  • Diploids: 2 pairs of homologous chromosomes → 2 chromosomes that have the same genes

In humans:

• Haploid number of chromosomes

• Diploid number of chromosomes

• Haploid number of chromosomes: 23

• Diploid number of chromosomes: 46

What are the parts of a metaphase chromosome?

• Telomeres:

  • Tandem repetas of repetitive sequences

  • Provide stability

• Centromere:

  • Appears as a constriction

  • Drive chromosomic movements during cell division

• Chromatids:

  • Arms

How many genes do humans have? How many are protein-coding? How many are not?

• Approx 35 000 genes

• 20 000 protein-coding

• 15 000 non-protein-coding

What is a karyogram?

It is a visualization of chromosomes by staining

In what phase do the chromosomes usually are in a karyogram?

In the metaphase

What is the banding pattern in this staining technique?

G-banding

• Dark bands are AT rich

• Pale bands are GC rich

The most common one

What is the banding pattern in this staining technique?

R-banding

• Dark bands are GC rich

• Pale bands are AT rich

What is the banding pattern in this staining technique?

Q-banding

• Dark bands are AT rich

• Pale bands are GC rich

(Different staining procedure than G-banding)

What is the banding pattern in this staining technique?

C-banding

• Dark bands contain constitutive heterochromatin → to stain centromeres

Which types of chromosomes are there?

1. Telocentric

2. Acrocentric

3. Sub-metacentric

4. Metacentric

Which type of chromosome is it?

Sub-metacentric

Which type of chromosome is it?

Telocentric

Which type of chromosome is it?

Acrocentric

Which type of chromosome is it?

Metacentric

True or false: the X and Y chromosome are the same size

False: they are not homologous

• X: submetacentric medium chromosome

• Y: small acrocentric chromosome

What are the characteristics of sex chromosomes?

• Heteropyknotics: they stain differently than other chromosomes

• Delay in duplication

• Delay in ordering

Chromosome band numbering → what does each letter/number refer to?

1p22.1

• 1: chromosome

• p: arm of the chromosome

  • Short arm (p)

  • Long arm (q)

• 2: region

• 2: band

• 1: subband

What is the karyotypes’ function?

They provide structural organization of individual genomes → you can detect deletions, insertions, duplications, translocations of large DNA fragments (you can detect big mutations)

What can you say about the patient just seeing the karyotype?

• Aneuploidy (abnormal number of chromosomes): trisomy of chromosome 21

• 2 X chromosomes: female

True or false: the GC content varies among species, across genomes and it is not uniform in a same chromosome

True

• Eukaryotes have low range of variation: 35-45%

• Prokaryotes have much wider range: 25-70%

What is an example of the % of GC content varying within a chromosome?

• CpG islands: genomic region with high frequency of CG dinucleotides relative to the rest of the genome → (GCGCGCGCGC)n

• This is very typical in the upstream promotor sequences → methilation occurs in C: the gene won’t be expressed

True or false: humans have low repetitive DNA

False, almost 50% is repetitive

How can it be shown that a high % of genomic DNA is repetitive?

Through denaturing/renaturing experiments: repetitive sequences associate and dissociate much quicker, while non repetitive DNA takes longer

if the DNA is repetitive, when you do the hybridazion step, it will hybridaze much quicker (más concentración de esa secuencia, tendrá más probabilidad de encontrarse)

What are the two types of repetitive DNA?

• Interspersed repeats: repeticiones intercaladas → most repetitive sequences are interpersed repeats

• Tandemly repeated DNA: repeticiones seguidas, pequeños grupos donde no hay otras secuencias entre medio

How can the human genome be classified?

• 1/3 of the genome: genes and gene-related sequences (1200 Mb)

  • Exons (48 Mb): codificant region of the gene

  • Related sequences (1152 Mb):

    • Pseudogenes: nonfunctional genomic DNA sequences that closely resemble functional protein-coding genes but have lost their ability to encode proteins due to accumulated mutations

    • Gene fragments

    • Introns, UTRs

• 2/3 of the genome: Intergenic DNA (2000 Mb) → basically repetitive DNA

  • Interpersed repeats (1400 Mb):

    • LINE’s (640 Mb)

    • SINE’s (420 Mb)

    • LTR elements (250 Mb)

    • DNA transposons (90 Mb)

  • Tandemly repeats (600 Mb):

    • Microsatellites = simple sequence repeats (SSR) (90 Mb)

    • Various (510 Mb)

Which DNA content corresponds to these %?

• 8%

• 3%

• 13%

• 20%

• 3%

• 5%

• 8%

• 12%

• 26%

• 1,5%

• Interpersed Repeats → 44%

  • 8% → LTR retrotransposons

  • 3% → DNA transposons

  • 13% → SINE’s

  • 20% → LINE’s

• 3% → SSR (microsatellites)

• 5% → segmental duplication

• 8% → miscellaneous (diverse) heterochromatine (condensed)

• 12% → miscellaneous unique (not repetitive) sequences

• 26% → introns (inside the gene)

• 1,5% → protein coding

What is the importance of identying repetitive DNA?

• Repetitive DNA influences the structure and function of the genome (chromosome rearrangement, transcriptional regulation)

  • Repetitive DNA: usually condensed → heterochromatin: gene silencing

  • Unique DNA: usually relaxed → euchromatin: gene expression

• Importance in disease: recombination events resulting in duplications or deletions

What types of repetitive DNA are there?

• Interspersed repeats (transposon-derived repeats)

• Processed pseudogenes → An mRNA transcript from a functional gene is accidentally reverse-transcribed into DNA by an enzyme (usually from a LINE element) and then inserted back into the genome.

• Simple sequence repeats

• Segmental duplications

• Blocks of tandemly repeats sequences

Repetitive DNA: how are interpersed repeats (trasposon-derived repeats) generated?

• They can be generated by:

  • Copying a DNA intermediate → DNA transposons

    • A transposable gene is flanked by two inverted repeats (IR)

    • A transposase will recognize the IR and cut and paste the gene in another part of the genome

  • Copying a RNA intermediate → retroelements (RNA intermediate) = retrovirus, retrotransposon

    • A retrotransposon will go through transcription and generate a single-stranded RNA

    • It will go through reverse transcription and generate double-stranded DNA → cDNA: it doesn’t contain UTR and introns

    • It will then reintegrate in the genome randomly (copy and paste) → they are highly replicative, that is why they are highly abundant in the genome

Repetitive DNA: how much of the human genome interpersed repeats do represent?

45% approx

What are long terminal repeats (LTR)?

DNA sequences found flanking retrotransposons and retrovirus (common to viral retroelements)

Repetitive DNA: what are the 4 types of interpersed repeats in humans?

• Retrotransposons:

  • Long interpersed nuclear elements (LINE): 20%

    • Autonomous non-LTR retrotransposons

    • Encode a reverse transcriptase

  • Short interpersed nuclear elements (SINE): 13%

    • Non-autonomous non-LTR retrotransposons

  • Long-terminal repeat (LTR) retrotransposons: 8%

    • Autonomous LTR retrotransposons

    • Encode a reverse transcriptase

• Transposons:

  • DNA transposones: 3%

    • Autonomous

    • Encode a transposase

Repetitive DNA: Are interpersed repeats present in eukaryotic genomes?

Yes

Repetitive DNA: what are pseudogenes?

A nonfunctional gene copy

Repetitive DNA: what are conventional pseudogenes?

An initial mutation inactivates the function

Repetitive DNA: what are processed pseudogenes?

• They arise by abnormal retrotransposition event on a functional gene → chat: An mRNA transcript from a functional gene is accidentally reverse-transcribed into DNA by an enzyme (usually from a LINE element) and then inserted back into the genome.

• They lack introns and is inactive (no upstream regulatory sequences, no expression)

Repetitive DNA: what are sinple sequence repeats (SSR)?

They are tandemly repeated DNA

• Minisatellites: n repeats lager than 13 bp (up to 500 bp) (clusters of up to 20 kb)

• Microsatellites: n repeats of up to 13 bp (clusters of up to 150 bp) → more abundant, that is why when we say SSR we usually refer to microsatellites

What is a polymorphism?

It is the presence of genetic variation wiithin a population, upon which natural selection can operate

Repetitive DNA: why microsatellites have a high mutation rate?

It is because of replication slippage: occurs at the repetitive sequences when the new strand miss pairs with the template strand → it causes microsatellite polymorphisms

During replication, DNA strands are opening and closing, because they are so similar, they can bind incorrectly, making a mismatch/loop

It can be a:

• Backward slippage: the new strand has the loop (addiotional nt) → we add a repetition

• Forward slippage: the template has a loop (the new strand will have fewer nt) → we lose a repetition

Repetitive DNA: why can microsatellites be used in genetic profiling of individuals?

• Because they are highly polymorphic elements → high variation and high heterozygosity

• For example, each microsatellite averages 10 allelic variants

Repetitive DNA: how microsatellites are used to identify individuals? Simple Sequence Lenght Polymorphisms (SSLPs analysis)

• The FBI have identified 13 different SSRs → at least everyone should have one (in 2017 they incremented it to 20)

• They have designed a set of 13 different primers corresponding to the SSRs → depending on the different combination of microsatellites each individual has, different bands will appear in the gel

• It will allow us to differentiate genomes

• D: victim

• A: suspect 1

• C: suspect 2

Who is the killer?

A, because the anorak swab matches

Repetitive DNA: what are segmental duplications (low copy repeats)?

• They are two genomic regions sharing >90% nt identity over a span of 1kb

• Common in plant and animal genomes

• 5% of genomic DNA are segmental duplications

• They make difficult the sequence assembly and the genetic dissection

• Unknown function other than redundancy

Compared with other mammals, the genomes of — and other — show an enrichment of large, interpersed — —.

• Humans

• Primates

• Segmental duplications

Repetitive DNA: what are blocks of tandemly repeated sequences?

• Occurs in telomeres and centromeres in the form of heterochromatin

  • Telomeres:

    • Human telomeric repeats: (TTAGGG)n

    • Telomere lenght:

      • 11 kbp at birth

      • < 4 kbp in old age

  • Centromeres:

    • a 1-4 Mb region with alpha-satellite repeats (171 bp repeats)

• They have high density beacuse they are very repeated → they are satellite bands in density gradient centrifugation of genomic DNA

True or false: gene density does not vary

False: it depends on the sequence

Gene content: what is the gene definition?

The one gene - one functional RNA hypothesis: the gene is a portion of DNA required for the expression of a functional gene product (an RNA or a protein)

Gene content: the — is the basic atomic unit of inheratance

Transcript

What is an open reading frame (ORF)?

The part of DNA or RNA that ha sthe potential to be translated into a protein

From start codon to stop codon

Gene content: what are the types of genes?

• Protein-coding genes:

  • Major category

  • Minimum ORF 90 bp (30 aa)

• Non-protein-coding genes:

  • tRNA → protein synthesis

    • Function: The "adaptor" molecule. It physically brings the correct amino acid to the ribosome during translation (protein synthesis). It reads the codon on the mRNA and drops off the amino acid.

  • rRNA → protein synthesis

    • Function: The "factory floor." It is the main structural and catalytic component of the ribosome (the machine that builds proteins). The ribosome is actually made of rRNA and proteins.

  • snoRNA: small nucleolar RNA → rRNA processing

    • Function: "rRNA processing." These work in the nucleolus (a part of the nucleus) to chemically modify and cut up rRNA molecules to help them mature into functional ribosomes.

  • snRNA: small nuclear RNA → splicing

    • Function: "splicing." These are the main components of the spliceosome. The spliceosome is the complex that cuts out introns from pre-mRNA and joins the exons together to make the mature mRNA.

  • miRNA: microRNA → regulation

    • Function: "regulation." These are short RNAs (about 22 nucleotides) that bind to specific messenger RNAs (mRNAs) and block them from being translated or mark them for destruction. They are a key part of gene regulation (turning genes off).

  • lnc-RNA: long non-coding RNA

    • Function: A catch-all category for long RNA molecules (longer than 200 nucleotides) that don't code for protein. They do a huge variety of jobs, including silencing entire chromosomes (like X-chromosome inactivation) and controlling transcription.

What are ESTs?

• Expressed sequence tags

• Short sequence of DNA that is generated by sequencing one or both ends of a cDNA clone

• They are a subtype of cDNA library but not complete

Gene content: how can we identify exons?

By alignments of cDNA and EST to the genome

Gene content: we use extrinsic (BLAST) and intrinsic (DNA patterns such as start and stop codon) algorithims to define:

• Intron/exon boundaries (GT/AT) → GT al inicio del intrón y AT al final (splicing sites)

• Exons (noncoding 5’ and 3’ UTR, ATG, Stop)

• Regulatory elements (basal-TATA-box, proximal, distal)

Gene content: more complex → — exons

Give an example of a complex gene:

• more

• Titin (connectin, muscle elasticity): has 364 exons

Gene density: what does it mean to have a compact genome? Give an example of an organism that has a compact genome and an organism that has a non-compact genome:

• To have a high number of protein-coding-genes and a low number of repetitive DNA

• Compact genome: yeast

• Non-compact genome: maize

Gene content: what does the Gene Ontology classify?

It classifies the molecular function, the biological process and the cellular component of a gene

Gene content: why non-protein-coding genes (ncRNA) are more difficult to identify in a genome than exons?

• They are more difficult to predict

  • Not represented in cDNA libraries (no polyA)

  • Conserved the secondary structure (not codons): no sequence similarity searches

    • La función depende de cómo se pliega el ARN, no de qué nucleótidos exactos tiene. La secuencia puede cambiar mucho siempre que los pares de bases que forman la estructura se mantengan. La secuencia cambió, pero la estructura sigue igual → la función se conserva → la evolución lo permite. BLAST busca similitud de secuencia, no de estructura.

• Less known function and distribution

Regulatory regions: what are cis-regulatory modules (CRMs)?

They are promotors, enhancers and silencers → no tienen una secuencia fija, por eso necesitamos métodos experimentales para identificarlas

Regulatory regions: what is an experimental tool that can be used for identification of cis-regulatory modules (CRMs)?

Chromatin Immunoprecipitation: ChIP-seq or ChIP-chip → where are transcription factors or histones bound in the genome?

1. DNA-protein cross linking: stabilizes DNA interacction

2. Cell lysis: free DNA and proteins

3. DNA fragmentation by sonication or enzyme digestion

4. Inmunoprecipitation:

   1. add an specific antybodu against a transcription factor or a histone modification

5. DNA purification: you eliminate the protein and only save the DNA that was bound to the antibodies

6. You sequence these segments

Regulatory regions: how can CpG islands determine gene expression or silencing?

CpG islands: high proportion of C-G

• When not methylated: active promotor → gene expression

• When methylated:

  • C-methylation

  • Recruitment of Methyl-CpG-binding proteins (MeCP)

  • Recruitment of Histone-deacetylase (HDAC): they remove acetyl group of hystones, now they are + charged and bind more to DNA (-)

  • Chromatin will be more compact → gene silencing