KENYATTA UNIVERSITY

SBC 312: Genome Organization IIDepartment of Biochemistry

& Biotechnology

Dr. P. OjolaSemester-I 2017/2018

Course outline: Higher order structure in chromatin, organization of

chromatin in the cell nucleus, Heterochromatin and Euchromatin,

Scaffolds and Dormains. The nuclear matrix, The boundary of

eukaryotic Nucleus: The nuclear envelope structure, Transport through

the nuclear envelop, Nuclear structure in Prokaryotes, bacterial

nucleoid structure, bacterial nucleiod proteins, Nucleiod Structure in

Cyanobacteria, Dinoflagellates; Eukaryotes with Prokaryotic DNA

organization

Reference: Genes VIII Benjamin Lewin, Genetics. Analysis of genes

and genomes 5th Ed. Daniel L. Hartl and Elizabeth W. Jones

-1870-nucleus plays a key role in inheritance,

-chromosomes 1st observed inside nucleus as threadlike objects

-No. of chromosomes in each cell differs among biological species,

-but number of chromosomes in nearly always constant within cell

particular species

- human genome, contained chromosomes of a sperm or egg, Is ~3

billion nucleotide pairs of DNA.

.

Introduction

• genome – The complete set of sequences in the

genetic material of an organism.

– It includes the sequence of each chromosome plus

any DNA in organelles.

– sexual organisms, genome is usually regarded as

DNA present in a reproductive cell

• transcriptome – The complete set of RNAs

present in a cell, tissue, or organism.

– Its complexity is due mostly to mRNAs, but it also

includes noncoding RNAs.

Introduction

• proteome – The complete set of proteins that is

expressed by the entire genome.

– The term is sometimes used to describe the

complement of proteins expressed by a cell at any

one time.

• interactome – The complete set of protein

complexes/protein–protein interactions present

in a cell, tissue, or organism.

- eukaryotes DNA has unique and repeated sequences.

-~5% of human DNA encodes proteins fxnal RNAs and regulatory sequences

-remainder merely spacer DNA

-this DNA, ~50% in humans, derived from transposons, genetic symbiots that have

contributed to the evolution of contemporary genomes.

-Each chromosome has a single,long molecule of DNA up to ~280 Mb in humans,

-These organized into increasing levels of condensation by the histone an nonhistone proteins

- Much smaller DNA molecules are localized in mitochondria and chloroplasts

Chromosome Organization and Molecular Structure

Chromosomes & Genomes

• Chromosomes

– complexes of DNA and proteins – chromatin

– Viral – linear, circular; DNA or RNA

– Bacteria – single, circular

– Eukaryotes – multiple, linear

• Genome

– The genetic material that an organism possesses

– Nuclear genome

– Mitochondrial & chloroplasts genome

• Genome is Infectious particles containing nucleic

acid surrounded by a protein capsid

• Rely on host cell for replication, transcription,

translation

• Exhibit a limited host range

• Genomes vary from a few thousand to a

hundred thousand nucleotides

Viruses

Some Virus Structures

Phage

Capsid

protein

TMV

• In a region called the

nucleoid

• DNA in direct contact

with cytoplasm

Bacterial Chromosomes

Prokaryote genomes

● Example: E. coli

● 89% coding

● 4,285 genes

● 122 structural RNA genes

● Prophage remains

● Insertion sequence elements

● Horizontal transfers

• Eukaryotic species contain one or more sets of chromosomes (ploidy level)– Each set is composed of several linear chromosomes

• DNA amount in eukaryotic species is greater than that in bacteria

• Chromosomes in eukaryotes are located in the nucleus– To fit in there, they must be highly compacted

• This is accomplished by the binding of many proteins• The DNA-protein complex is termed chromatin

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Eukaryotic Chromosomes

• vary substantially in size

– variation not related to complexity of the

species

– i.e - a two fold difference in genome size

between two salamander species

• Size difference due to accumulation of

repetitive DNA sequences

Eukaryotic genomes

Eukaryotic genome

● Example: C. elegans

● 10 chromosomes

● 19,099 genes

● Coding region – 27%

● Average of 5 introns/gene

● Both long and short duplications

Evolution of genomes

● Adaptation of species is coterminous with

adaptation of genomes

● Where do genes come from? (Answer: from other

genes)

● Homologs and paralogs

● Lateral transfer

● Molecular species each have their own family tree

● Genes are widely shared

Close relatives

● Yeast, fly, worm and human share at least 1308

groups of proteins

● Unique to vertebrates: immune proteins (for

example)

● Unique molecules are adapted from ancient

molecules of different purpose but similar design

● Most new proteins come from domain rearrangement

● Most new species come from control region

variation

Figure 10.10

Variations in DNA Content

• Eukaryotic chromosomes are long, linear

DNA molecule

• Three types of DNA sequences are required

for chromosome replication and segregation

– Origins of replication (multiple)

– Centromeres (1)

– Telomeres (2)

Eukaryotic Chromosome Organization

Centromere

Kinetochore proteins

Origin of replication

Origin of replication

Origin of replication

Origin of replication

Telomere

Telomere

GenesRepetitive sequences

Chromosome Organization

• Genes located between centromere & telomeres

– hundreds to thousands of genes

– lower eukaryotes (i.e. yeast)

• Genes are relatively small

• Very few introns

– higher eukaryotes (i.e. mammals)

• Genes are long

• Have many introns

• Non-gene sequences

– Repetitive DNA

• Telomere

• Centromere

• Satellite

Eukaryotic Gene Structure

Promoter/

Enhancer

Cis-

Regulatory

Elements

Start Codon

ATG

Exon1 Exon2 Exon3

Stop Codon

TAA, TAG, TGA

• Sequence complexity refers to the number of

times a particular base sequence appears in

the genome

• 2 main types of sequences

– Moderately repetitive

– Highly repetitive (low complexity)

Repetitive Sequences

• Unique or non-repetitive sequences

– Found once or a few times in the genome

– Includes structural genes as well as intergenic areas

• Moderately repetitive

– Found a few hundred to a few thousand times

– Includes

• Genes for rRNA and histones

• Origins of replication

• Transposable elements

Repetitive Sequences

10-28

• Highly repetitive

– Found tens of thousands to millions of times

– Each copy is relatively short (a few nucleotides to several

hundred in length)

– Some sequences are interspersed throughout the genome

• Example: Alu gene family (repetitive transposable elements) in

humans

– Other sequences are clustered together in tandem arrays

• Example: centromeric satellite & telomeric regions

Repetitive Sequences

Gross structure of chromosomes

Kinetochore: not the same in all organisms. There are two main types;(1) trilaminar /stratified structure e.g many animals and lower plants;

(2) ball and cup structure e.g higher plants.

- trilaminar type consists of an outer dense layer 30–40 nm thick, - middle layer of low density 15–60 nm thick, inner dense layer, 15–40 nm thick,which is granular like the chromatin, and is dense and compact (Fig. 19.1

).

-The middle layer is structure-less, and has a clear area called the corona.

The kinetochore usually takes after the shape of the centromere where it lies. In some

elongated centromeres the kinetochore may be 1.4 nm long and only 0.4 nm wide.

-Its size is increased by spindle position.

-ball and cup type of kinetochore has a depression (the cup) about 1.5 μm across,

-And amorphous mass, the ball, ~0.8 μm across in the middle of the depression .

-The ball is attached to the bottom of cup, and the spindle microtubules attached all

round the sides of the ball.

-Diffuse centromere: Sometimes there is no localised centromere in a chromosome.

Such

holocentric chromosomes are present plants like Luzula, some members of Cyperaceae,

algae, protozoa and insects like Steatococcus and Tamalia.

-When such a chromosome is broken into small fragments by radiation, all the

fragments move independently to the poles.

Heterochromatin and euchromatin

Heitz (1928, 1933) distinguished two types of chromosome material—

heterochromatin- highly condensed throughout interphase,

Euchromatin-unravels at the end of mitosis and stains weakly in interphase nucleus.

-Brown (1966) suggested two distinct types of heterochromatin,

facultative and constitutive

-Facultative heterochromatin-present in one or the other of a pair of homologouschromosomes, not both.

E.g inactivation of one chromosome in mammalian females during early stages of development. In adults it is visible as the Barr Body in cells of buccal mucosa but absent in male cells

Constitutive heterochromatin is permanently condensed and is found in the same

locationsin both homologous chromosomes.

-It is often present in specific regions of chromosomes such as the centromeres (Fig. 19.2c), telomere, nucleolus organising regions and other secondaryconstrictions

Centromere function

-DNA is replicated during S (synthetic) phase of interphas.

-Spindle fibres bind to the centromere, and pull the sister chromatids apart to the two

poles

- Centromeres consist of specific DNA sequences where centromere-associated proteins

bind.

The DNA-protein complex is the kinetochore

-which molecular motors that drive movement of chromatids to the two poles

sequences required for centromere function

In Saccharomyces cerevisiae sequence elements, are two short sequences of 8 and 25 bp

separated by 78 to 86 bp of very AT-rich DNA.

-yeast, S. pombe sequences much larger, spanning 40 to 100 kb of DNA

-absence of a fnal centromere, the plasmid unable to segregate properly

-Drosophila centromere spans 420 kb, and ~ 85% consists of two highly repeated satellite DNAs having sequences AATAT and AAGAG

-The remainder consists of interspersed transposable elements, that may also be present at other sites in genome, and a nonrepetitive region of AT-rich DNA.

-Deletion of the satellite sequences, transposable elements and the nonrepetitive DNA reduced functional activity of the centromere

-hence kinetochore formation and centromere function compromised

-Mammalian centromeres has extensive heterochromatin regions that contain highly repeated satellite DNA sequences. Their precise function in mammalian cells is not known

Telomere

-a specialised structure at the extremity of a linear chromosome that is essential

for the maintenance of chromosome stability

-absence produced sticky ends and unstable chromosomes.

-contain sequences that play important roles in chromosome replication

-DNA sequences in telomeres are similar across a wide range of lower and higher eukaryotes, the same type of sequence is present in plants and humans

-. Each telomere consists of tandem arrays of highly repeated sequences of DNA containing clusters of G-C residues on one strand.

-Thus, the sequence repeats in humans and various mammals is 5′-TTAGGG-3′ and in Tetrahymena it is 5′-GGGGTT-3′.

-One unusual feature of the telomere sequence is extension of the G-C rich strand, by about 14 to 16 bases, as an overhanging tail of single-stranded DNA

-DNA replication cannot begin precisely at the 3, end of a template strand,

-therefore, the 3’end of replicated duplex DNA must terminate in a short stretch where

the DNA is single-stranded.

-The single-stranded overhanging DNA is subject to degradation by nucleases during

replication.

- If no mechanism is put to restore the end digested by nucleases, the DNA

molecule in a chromosome would become slightly shorter with each round of

replication

-restoring ends of a DNA molecule in a chromosome involves telomerase.

-first discovered in the ciliated protozoan Tetrahymena, adds tandem repeats of a simple

sequence—TTGGGG-3′ to the 3′ end of a RNA strand;

in humans this enzyme adds the sequence –TTAGGG-3′.

-The tandem repeats of these sequences constitute the telomere.

- As the repeating telomere sequence is being elongated, DNA replication takes place

resulting in synthesis of a partner strand.

-telomerase is remarkable by incorporating essential RNA molecule - guide RNA,

-that contains sequences complementary to the telomere repeat.

-guide RNA serves as a template for telomere synthesis and elongation.

-guide RNA undergoes base-pairing with the telomere repeat and serves as a template

for telomere elongation by the addition of more repeat units.

-Notably, the complementary DNA strand of the telomere is synthesised by the cellular

DNA synthesising enzymes

-the repeated sequences of telomeric DNA form loops at the ends of the chromosomes,

to protect the chromosome terminus from degradation

-The telomeric DNA is anchored to the

nuclear matrix by proteins called Ku

proteins.

-Ku an abundant nuclear protein.

-It exists as a heterodimer of two

subunits of 70 kDa and 80 kDa.

-Ku functions in capping the telomeres,

preventing chromosome end fusions,

and in telomere length control.

-Ku binds DNA ends in a sequence-

independent manner