The Eukaryotic Chromosome: An Organelle for Packaging and Managing DNA

Eukaryotic Chromosomes: A chromosome consists of a single double-helix DNA molecule starting at one end of the chromosome going through the centromere and ending at the other end of the chromosome. Chromatin consists of 1/3 DNA, 1/3 histones and 1/3 non-histones Histones are five types, H1, H2A, H2B, H3 and H4. They are the same in all cell types of an organism and in all different eukaryotic organisms. Histones are highly conserved basic proteins that form nucleosomes, a spool-like structure upon which 160 base pairs of DNA is wound. Linker DNA between nucleosomes is 40 base pairs long. Non-histones are all other types of proteins (enzymes included) that are responsible for DNA replication, expression and also cell division. These are very heterogeneous group of proteins.

Karyotypes: represent the metaphase chromosomes of a cell that are fully condensed then stained with Giemsa stain. This staining forms G bands which are interchangeable dark and light bands along the chromosome. These bands are identical and characteristic for each pair of homologous chromosomes but differ between different chromosomes. At low resolution, human chromosomes have 300 dark G bands and light interbands. At high resolution there are 2000 of such bands. Banding pattern of G bands is species specific. Bands are used to locate and map genes, especially useful when mapping disease-causing genes. For example the the X-linked gene for color blindness resides at q27-qter. Evolutionary relationships can be explained by G banding patterns. Chromosome 1 of great apes have are very similar G bands as those of chromosome 1 in humans. Chromosome 2 of humans appears to have resulted from the fusion of acrocentric chromosomes of apes.

G bands of human chromosomes have been used to identify genetic diseases. Missing of a light band in X chromosome was linked to the appearance of four X-linked diseases in a single individual.

Chromosome structure ensures accurate replication & segregation: 1. Origins of replication: 10,000 in mammalian cells scattered throughout the chromosomes and are separated by 30-300 kb of DNA. At any origin of replication, the replication occurs at both ends of the replication bubble (replication fork) in opposite directions. The DNA between two origins of replications is called a replicon or a replication unit. Origins of replication consist of an AT-rich sequence (consensus) that is adjacent to special flanking sequences. In yeast, DNA sequences containing origins of replication are isolated by their ability to replicate plasmids when incorporated into their DNA. Hence they are called autonomous replicating sequences (ARS).

Origins of replication sequences are not associated with nucleosomes and are accessible to enzymes. 2. Telomeres ensure that chromosomes do not lose their termini at each round of replication: DNA polymerase is unable to fill in an RNA primer’s length of nucleotides at the 5’ end of a new strand at chromosome tips. This results in shortening the ends of a chromosome, with all the relative genes it carries, a bit at a time with every round of DNA replication. Telomeres are 250-1500 repeats of the sequence TTAGGG at the ends of chromosomes. Such repeats have 2 functions: (i) form a hairpin loop by unusual G-G hydrogen bonding. This provides a free 3’ end for finishing the replication of the 5’ end of newly synthesized DNA strands. (ii) attract telomerase, a ribonucleoprotein which extends broken telomeres.

Centromeres: - are primary constriction in chromosomes which contain blocks of repetitive, noncoding DNA sequences known as satellite DNA - satellite DNA consist of short tandem repeats (5-300 base pairs long). In humans, a 171 bp satellite DNA is present in tandem repeats at the centromere region. - Centromeres have two functions. They hold sister chromatids together and ensure proper segregation of chromosome segregation (separation and distribution) through their kinetochore region with motor proteins that specifically bind to microtubules of the spindle apparatus. - In yeast, centromeres consist of two highly conserved sequences each 10-15 bp separated by 90 bp of AT-rich DNA. Higher eukaryotes have larger and more complex centromeres. Yeast artificial chromosomes (YAC) demonstrate the important elements for chromosome function.

Chromosome structure & Gene Expression: - Gene expression occurs in the interphase. Decompaction of higher folding precedes transcription. - RNA polymerase unwinds the nucleosome and proceeds in the direction 5’ to 3’. DNA left behind the polymerase during transcription rewinds again around histones to form nucleosomes. - DNase I treatment experiments showed that DNase Hypersisitive sites (DH) that contain few nucleosomes are found at the 5’ end of genes. - Extreme condensation causes the formation of heterochromatin which could be constitutive or facultative. - Position effect in Drosophila is an example of facultative heterochromatin and Barr bodies in humans are constitutive heterochromatin

How chromosomal packaging influences gene activity Decompaction precedes gene expression Boundary elements delimit areas of decompaction Nucleosomes in the decompacted area unwind to allow initiation of transcription Transcription factors (nonhistone proteins) unwind nucleosomes and dislodge histones at 5’ end of genes Unwound portion is open to interaction with RNA polymerase which can recognize promotor and initiate gene expression

Studies using DNase identify decompacted regions Figure 12.12 a Fig. 12.12 a

Extreme condensation silences expression Heterochromatin Darkly stained region of chromosome Highly compacted even during interphase Usually found in regions near centromere Constitutive heterochromatin remains condensed most of time in all cells (e.g., Y chromosomes in flies and humans) Euchromatin Lightly stained regions of chromosomes Contains most genes

Heterochromatin versus euchromatin Heterochromatin is darkly stained Euchromatin is lightly stained C-banding techniques stains constitutive heterochromatin near centromere Figure 12.13 Fig. 12.13

Facultative heterochromatin Position effect variegation in Drosophila: moving a gene near heterochromatin prevents it expression Facultative heterochromatin Moving a gene near heterochromatin silences its activity in some cells and not others Figure 12.14 a Fig. 12.14 a

A model for position-effect variagation Position effect variegation in Drosophila: moving a gene near heterochromatin prevents it expression A model for position-effect variagation Heterochromatin can spread different distances in different cells Figure 12.14 b Fig. 12.14 b

Barr bodies: example of heterchromatin decreasing gene activity Barr bodies – inactivation of one X chromosome to control for dosage compensation in female mammals One X chromosome appears in interphase cells as a darkly stained heterochromatin mass

Unusual chromosome structures clarify the correlation between chromosome packaging and gene function Polytene chromosomes magnify patterns of gene expression and tie them to gene expression Drosophila larvae salivary gland cells – chromosomes replicate 10 rounds without mitosis 210 = 1024 sister chromatids plus homolougous chromosome tightly wound together – 2048 double helices of DNA Chromosomal puff of gene activity Figure 12.15 a Fig. 12.15 a

Darkly stained highly condensed bands interspersed with lightly stained less condensed bands Figure 12.15 b Fig. 12.15 b

Polytene chromosomes are an invaluable tool for geneticists in situ hybridization of white gene to a single band (3C2) near the tip of the Drosophila X chromosome Figure 12.15 c Fig. 12.15 c

Nucleolus contains hundreds of copies of rRNA genes Decondensed chromatin in the nucleolus of interphase cells contains rRNA genes actively undergoing transcription Figure 12.16 a Nucleolus contains hundreds of copies of rRNA genes Fig. 12.16 a

Newly transcribed rRNAs appear as short, wispy strands emanating from the DNA in this electron micrograph of nucleolus chromatin Figure 12.16 b Fig. 12.16 b