Det humane genom                                Menneskekroppen har 100 billioner celler, hver med 46 kromosomer. Samlet lengde av DNA: 2 meter/celle

…er ganske stort

Andre genomer som kan lære oss mye om det humane genom

Antall kromosomer i forskjellige organismer                             

Ophioglossum reticulatum K-value paradox: Complexity does not correlate with chromosome number. Homo sapiens Lysandra atlantica Ophioglossum reticulatum 46 250 1260

Størrelse av genomer

C-value paradox: Complexity does not correlate with genome size.

Det humane genom

The human genome is disappointing: It is small It is empty It is unoriginal It is repetitive

En oversikt over det humane genom

Hvor mange gener i genomet?

Genomstørrelser – hvor mange gener?

N-value paradox: Complexity does not correlate with gene number. ~31,000 genes ~26,000 genes ~50,000 genes

Sammensetning av genomet

1.5% The genome is empty. Exons Introns (junk) Intergenic regions

Den molekylære funksjonen til 26383 humane gener

Funksjonelle kategorier i eukaryote proteomer

Flere proteiner fra samme gen (alternativ spleising) Menneske: 60 % av genene koder for mer enn ett protein Orm: 22 % av genene koder for mer enn ett protein

Forskjeller i geninhold Fibroblastvekstfaktor – menneske 30, bananflue og orm 2 hver Transformerende vekstfaktor β – menneske 42, bananflue 9, orm 6 Gener som koder for proteiner med immunglobulindomener – menneske 765, bananflue 140, orm 64 ”Sinkfinger”-proteiner – menneske dobbelt så mange som bananflue og 5 ganger flere enn orm

Mouse-human synteny. Human chromosomes can be cut into ~150 pieces, then shuffled into a reasonable approximation of the mouse genome.

CpG-frekvens og CpG-øyer The typical density of CpG doublets in mammalian DNA is ~1/100 bp, as seen for a -globin gene. In a CpG-rich island, the density is increased to >10 doublets/100 bp. The island in the APRT gene starts ~100 bp upstream of the promoter and extends ~400 bp into the gene. Each vertical line represents a CpG doublet.

CpG-øyer Diagram showing the structure of three human CpG island genes of different sizes. Vertical lines show the positions of CpGs in the first 10 kb of (a) the desmin (EMBL hsdes01), (b)hypoxanthine phosphoribosyl transferase (HPRT; EMBL hshprt8a) and(c) retinoblastoma (EMBL L11910) genes. The locations of the exons are shown by boxes. Open and tinted portions denote translated and untranslated regions, respectively. Any exons not present in the first 10 kb of genomic DNA are shown fused together to the right. The total genomic length of each gene (in kb) is given in brackets

Vedlikeholdsmetylering Ved maintenance-metylering induserer metyleringsmønsteret i en parental DNA-tråd det tilsvarende metyleringsmønster i den komplementære tråden. Slik kan et stabilt metyleringsmønster opprettholdes i en cellelinje

CpG – underrepresentert i genomet The CpG doublet occurs in vertebrate DNA at only ~20% of the frequency that would be expected from the proportion of G·C base pairs. (this is because CpG doublets are methylated on C, and spontaneous deamination of methyl-C converts it to T, introducing a mutation that removes the doublet.) In certain regions, however, the density of CpG doublets reaches the predicted value; in fact, it is increased by 10× relative to the rest of the genome. The CpG doublets in these regions are unmethylated

Cytosin, metylcytosin og tymin

Repetitive DNA I interspersed in tandem Alus are like that!

Repeterte sekvenser skaper problemer                                                          

Klasser av intersperserte repetisjoner i det humane genom

Elementer i det humane genom som kan transposeres på en RNA-formidlet måte RNA-mediated transposable elements in the human genome. Each contains the characteristic flanking direct repeats (arrows). The human endogenous retrovirus containing long terminal repeats (LTRs) (speckled region), gag (group-specific antigen gene), pol (polymerase gene) and env (envelope gene). The THE-1 retrotransposon consists of an open reading frame (ORF) and LTRs. The non-LTR retrotransposon (LINE) contains internal RNA polymerase II promoter sequences (P), two open reading frames, and an A-tail. The Alu element has a dimeric structure of homologous halves separated by a middle A-rich region (striped). The left half contains A- and B-box RNA polymerase III promoter sequences, and the right half contains an additional internal 31 bp. Other shaded regions are sequences unique to the element.

SINEs og utledning av fylogenetiske forhold En SINE er enten der eller ikke SINEs innsettes på tilfeldig måte i ikke-kodende områder. Samme plassering i to arter tyder på at innsettingen foregitt i en felles stamfar Innsetting av en SINE er irreversibel, fravær er derfor et ancestralt trekk

Alu elements Length = ~300 bp Repetitive: > 1,000,000 times in the human genome Constitute >10% of the human genome Found mostly in intergenic regions and introns Propagate in the genome through retroposition (RNA intermediates).

Evolution of Alu elements

Alu elements can be divided into subfamilies The subfamilies are distinguished by ~16 diagnostic positions.

Sekvenssammenstilling av Alu-familier 14 Alu-familier hos mennesket, hvorav 1 ikke hos andre primater. 2000 Alu-insersjoner spesifikke for mennesket. J, S, Y Alignment of Alu-subfamily consensus sequences. The consensus sequence for the Alu Sx subfamily is shown at the top, with the sequences of progressively younger Alu subfamilies underneath. The dots represent the same nucleotides as the consensus sequence. Deletions are shown as dashes, and mutations are shown in coloured boxes; all are colour-coded according to the family in which the ancestral mutation arose. Each of the newer subfamilies, such as Ya5 or Yb8, has all the mutations of the ancestral Alu elements, as well as five or eight extra mutations, respectively, that are diagnostic for the particular Alu subfamily. This figure primarily illustrates the newer subfamilies and does not attempt to show many of the older Alu subfamilies.

Evolusjon av Alu-elementer

Transposisjonering av et typisk humant Alu-element The structure of each Alu element is bi-partite,with the 3′ half containing an additional 31-bp insertion (not shown) relative to the 5′ half. The total length of each Alu sequence is ~300 bp, depending on the length of the 3′ oligo(dA)-rich tail. The elements also contain a central A-rich region and are flanked by short intact direct repeats that are derived from the site of insertion (black arrows). The 5′ half of each sequence contains an RNA-polymerase-III promoter (A and B boxes). The 3′ terminus of the Alu element almost always consists of a run of As that is only occasionally interspersed with other bases (a). Alu elements increase in number by retrotransposition — a process that involves reverse transcription of an Alu-derived RNA polymerase III transcript.As the Alu element does not code for an RNA-polymerase-III termination signal, its transcript will therefore extend into the flanking unique sequence (b). The typical RNA-polymerase-III terminator signal is a run of four or more Ts on the sense strand,which results in three Us at the 3′ terminus of most transcripts. It has been proposed that the run of As at the 3′ end of the Alu might anneal directly at the site of integration in the genome for target-primed reverse transcription (mauve arrow indicates reverse transcription) (c). It seems likely that the first nick at the site of insertion is often made by the L1 endonuclease at the TTAAAA consensus site. The mechanism for making the second-site nick on the other strand and integrating the other end of the Alu element remains unclear.A new set of direct repeats (red arrows) is created during the insertion of the new Alu element (d).

Alu-elementer hos primater

Eukaryotic genes (exons & introns) Splicing Translation

Alternative splicing: One gene, several proteins! Mature splice variant II variant I

Types of alternative splicing

Cassette exon or internal-exon skipping

Signals of splicing 1 2 Donor site Acceptor site Branch point YYYYYYYYYNCAG GTRAGT A CAG G Donor site Acceptor site Branch point Pyrimidine tract 1 2 A -OH Lariat A 1 2

Because mRNAs and Alus are frequently reverse transcribed and incorporated into the genome, pyrimidine tracts are ubiquitous The complementary strand of polyA is polyT = pyrimidine tract.

The minus strand of Alu elements contains “near” splice sites The minus strand of Alu contains ~3 sites that resemble the acceptor recognition site: Consensus acceptor site:YYYYYYNCAG/R Alu-J: (127-114) :TTTTTTGtAG/A The minus strand of Alu contains ~9 sites that resemble the consensus donor site: Consensus donor site: CAG/GTRAGT Alu-J: (25-17) : CAG/GTGtGA

 all Alu-containing exons are alternatively spliced. Our findings Out of 1,182 alternatively spliced cassette exons, 62 have a significant hit to an Alu sequence. Out of 4,151 constitutively spliced exons, none has a significant hit to an Alu sequence.  all Alu-containing exons are alternatively spliced.

Retention Ratio Retention ratio = number of mRNA molecules containing the alternatively spliced exon divided by total number of mRNA molecules. Retention ratio for Alu-containing exons was ~10%. Retention ratio for alternatively spliced exons that do not contain Alu was ~45%.

Proposed model for Alu exonization

Hvordan studere genomet? http://www.genome.ucsc.edu/cgi-bin/hgGateway?org=human http://www.ensembl.org/ Men NCBI har også en genombrowser: MapView!