Organisation of Eukaryotic Genome

Genome

• genome: genetic material of an organism or virus; the complete complement of an organism’s or virus’s genes along with its non-coding nuclei acid sequences (coding + non-coding)

• all organisms have a genome comprised of genetic material (DNA or RNA) that contains all genetic information needed to direct the development and maintenance of that organism

  • the entire complete set (or complement) of genetic information for all the proteins and RNA that the organism will ever synthesise

  • most genomes including the human genome and those of all other cellular life forms are made of DNA

  • viruses may contain DNA or RNA genomes

• prokaryotes - genome residues usually in a single DNA molecule; eukaryotic genome is much larger (human genome contains more than 5x no. of genes of a typical prokaryote)

• eukaryotic cells have a nuclear genome and a mitochondrial genome

  • for plants and algae - chloroplast genome

Eukaryotic genome

• biological information contained in a genome is encoded in its DNA and is functionally divided into discrete units called genes

• gene: a gene is a section of the DNA that contains the information in the form of a specific sequence of nucleotides/base to direct the synthesis of one polypeptide chain or RNA

  • unit of inheritance located in a fixed position (locus) on the chromosome which specifies a particular character of an organism

1. Genes are carried on chromosomes

• each gene resides in a specific location along the chromosome called the gene locus

2. Most eukaryotic genes are distributed among a species-specific number of linear chromosomes

• recall: DNA structure and replication

  • each chromosome is composed of a single DNA molecule (double helix) packaged with various histone and nonhistone proteins (e.g. scaffold proteins) found in the nucleus

  • a small proportion of eukaryotic DNA is found in the mitochondria

  • in the case of photosynthetic organisms, DNA is also found in the chloroplasts

3. Every eukaryotic cell has a complete copy of the nuclear genome

• each cell nucleus (except gametes) contains 2 sets of chromosomes, one from each parent - diploid cells

• either set of chromosomes is known as haploid set of chromosomes

• a compete eukaryotic genome comprises

  • one complete copy of genetic information carried by a haploid set of linear chromosomes in the nucleus (nuclear genome)

  • mitochondrial genome (consists of a single small circular DNA molecule)

  • chloroplast genome (in photosynthetic organisms only) which is also composed of one small circular DNA molecule

4. The human nuclear genome

• in humans, a complete copy of the nuclear genome comprises of ~3 × 10^9 DNA nucleotide base pairs distributed over 22 different autonomies and one of the two sex chromosomes (X or Y chromosomes)

• human genome sequence refers to the complete nucleotide sequence of DNA in these 23 chromosomes

  • diploid - a human somatic cell (i.e. cell that is not a germ cell or gamete) contains about twice this amount of DNA

human karyogram

The Complexity of the Eukaryotic genome

a. More complex organisms tend to have larger genome sizes

1. Genome size is usually expressed as the total number of base pairs (by) per haploid genome

• usually expressed in kilobases (kb) for base pairs in thousands or megabases (Mb) base pairs in millions

2. More complex organisms tend to have larger genome sizes compared to simpler organisms (e.g. prokaryotes)

• there is a correlation between an organism’s genome size and its apparent biological complexity because more genes and gene products are required to direct the development and maintenance of more complex organisms

3. Gene size is also larger in more complex organisms due to the increase in proportion of regulatory sequences needed for more complex control of gene expression (e.g. alternative splicing)

note: prokaryotes tend to have only one chromosome and a significantly smaller genome; also lack many regulator sequences present in eukaryotes

• e.g. in fig 5, genome size increases linearly with complexity of eukaryotes

• but this correlation is not observed between higher eukaryotes

Complexity of eukaryotic genome

b. no correlation between biological complexity of an organism and number of genes in its genome

1. Genome size is not necessarily proportional to number of genes in the genome

• e.g. humans have greater genome size but fewer genes than Pufferfish

2. There are other mechanisms at play to generate high biological complexity from a limited pool of genes

• action of different regulatory proteins interacting with specific regulatory elements to alter gene expression

Complexity of eukaryotic genome

c. Prokaryotic genomes have much higher gene densities than that of eukaryotes

1. the measure of the number of genes per million base pairs (Mb) in the genome —> gene density

2. human genome has an estimated 100-fold lower gene density than that of a typical prokaryote, despite its approximately 1000 times larger genome size

Complexity of eukaryotic genomes

d. The more complex eukaryotes generally have lower gene density than lower eukaryotes (higher eukaryotes = lower gene density)

1. decreased gene density especially in higher eukaryotes attributed to the large proportion of non-coding intergenic DNA relative to genes present in their genomes

• sequences are less compact and don’t belong in genes

• don’t encode any expressed protein or RNA product

note: prokaryotes typically have 85-90% of their genomes containing structural genes as compared to only 1-5% for the eukaryotic genome

Overview of DNA in eukaryotic genome

Packing of DNA in eukaryotic chromosome

First level of condensation (nucleosome fibre) —> Second level of condensation (solenoid) —> Third level of condensation (chromosomes)

First level of condensation

1. Nucleosomes packing process involves a molecule of DNA coiled around an octamer of histone proteins, two each of histones H2A, H2B, H3 and H4

• histones are small proteins with a high concentration of positively-charged residues e.g. lysine and arginine, which form ionic bonds with the negatively-charged sugar-phosphate backbone of DNA

• histones assemble into an octomer (8 histones) to form a core upon which 146 base pairs of DNA is bound

• double-stranded DNA is coiled around the histone core, forming a nucleosome core (gives chromatin a ‘beads-on-a-string’ look

• completed chromatin subunit consists of the nucleosome core, the linker DNA and the associated non-histone chromosomal proteins

2. Multiple nucleosomes are packed together to produce the 10-nm chromatin fibre also known as nucleosome fibre

First level of condensation

1. Nucleosomes packing process involves a molecule of DNA coiled around an octamer of histone proteins, two each of histones H2A, H2B, H3 and H4

• histones are small proteins with a high concentration of positively-charged residues e.g. lysine and arginine, which form ionic bonds with the negatively-charged sugar-phosphate backbone of DNA

• histones assemble into an octomer (8 histones) to form a core upon which 146 base pairs of DNA is bound

• double-stranded DNA is coiled around the histone core, forming a nucleosome core (gives chromatin a ‘beads-on-a-string’ look

• completed chromatin subunit consists of the nucleosome core, the linker DNA and the associated non-histone chromosomal proteins

2. Multiple nucleosomes are packed together to produce the 10-nm chromatin fibre also known as nucleosome fibre

Third level of condensation

1. Non-histone proteins known as scaffold proteins are involved in condensing the 30-nm chromatin fibre to form looped domains

2. In mitotic and meiotic chromosomes, the looped domains themselves coil and fold, further compacting all the chromatin to produce the characteristic metaphase chromosome

• width of one chromatid is 700nm

note: particular genes always end up located at the same places in mitotic and meiotic chromosomes, indicating that the packing steps are highly specific and precise

Role of condensation

• to organise and pack the giant DNA molecules of eukaryotic chromosomes into structures that will facilitate their segregation onto daughter nuclei

• DNA molecules of different chromosomes will not be entangled and as a consequence, break during separation at anaphase (prevent breakage)

Organisation of eukaryotic genome

1. Eukaryotic genomes comprise of coding (i.e. genes) and non-coding DNA sequences

• a eukaryotic gene includes not only the coding sequences that encode a functional gene product, but also non-coding regulatory nucleotide sequences required for proper expression of the gene

2. Total number of genes represented in the human genome is estimated to be approximately 1%; remaining DNA is non-coding

ref. to fig

(A) human chromosome 22, shown in its replicated form consisting of 2 chromatids joined at the centromere

(B) a ten-fold expansion of a portion of chromosome 22, with about 40 genes (dark regions) interspersed with intergenic DNA white regions)

(C) an expanded portion of (B) shows the entire length of 4 genes (dark regions), separated by intergenic DNA (gray regions)

(D) close-up of a gene in (C) showing the arrangement of non-coding introns (gray regions), coding exons (dark regions) and regulatory DNA sequences characteristic of a eukaryotic gene

organisation of eukaryotic gene at DNA level flowchart

Organisation of eukaryotic gene at DNA level

1. Eukaryotic protein-coding gene

• eukaryotic protein-coding gene requires the following DNA sequences for the proper expression of the gene

a. coding exons and non-coding introns —> transcription unit

b. non-coding DNA regulatory sequences

(see image)

2. Transcription unit

• as exons are interrupted by introns, exons are described as discontinuous coding DNA sequences of eukaryotic gene

• each exon codes for a particular portion (amino acid sequence) of the polypeptide while introns are not represented in the amino acid sequence of the protein gene product (i.e. introns are non-coding DNA sequences)

• number and sizes of introns per gene varies

• the amount of DNA in the intron sequences is often greater than the exons

note: prokaryote genomes are arranged in operons where multiple genes are clustered together under the control of a single promoter and regulatory region; no introns present and post-transcriptional modifications are not necessary prior to translation

3. Non-coding DNA regulatory sequences

• Regulatory sequences: regions of DNA sequence where gene regulatory proteins bind to control the rate of assembly of protein complexes required for gene expression

• regulatory sequences include

a. Promoter

• a series of DNA sequences located upstream of the transcriptional start site

• RNA polymerase and transcription factors bind to the promoter to initiate transcription

b. Control elements

• segments of DNA involved in regulating the initiation and rate of transcription by binding particular proteins

• include proximal and distal control elements that are located near to and far from the promoter respectively

i. Proximal control elements: sequences where gene regulatory proteins called general (or basal) transcription factors bind to initiate transcription

ii. Distal control elements consists of enhancers, DNA sequences that bind specific regulatory proteins known as activators to increase transcription rate, and silencers which interact with other specific regulatory proteins known as repressors to decrease transcription rate

enhancer (element) + activator (protein)

silencer (element) + repressor (protein)

note: elements —> DNA

factors —> proteins

c. Untranslated regions (UTRs)

• found in the exons of the mRNA but are not translated into polypeptide sequence

i. 5’ UTR

• starts at the +1 position on DNA template strand where transcription begins and ends one nucleotide before the start codon

• contains DNA sequence which is transcribed into a ribosome binding site on mRNA - ribosome binds to the mRNA and initiate translation

• contains DNA sequence which is transcribed into binding sites on mRNA for proteins which regulate the mRNA’s stability for translation

ii. 3’ UTR

• starts after the stop codon

• contains DNA sequence which is transcribed into a polyadenylation signal on mRNA, which is needed for termination of transcription

Organisation of the eukaryotic intergenic DNA

• these DNA sequences which are located between genes are termed intergenic DNA sequences consisting mainly of repetitive DNA

Repetitive DNA

• Repetitive DNA refers to sequences present in multiple copies in the genome

• Tandemly repeated DNA consist of DNA sequences repeated multiple times and arranged adjacent to one another in a head-to-tail fashion