Chromosome Structure and Function

Fundamental concepts and definitions

» A chromosome is a discrete DN A molecule which carries essential genetic information, together with any associated proteins which define its structure. The scope of the term can include the bacterial nucleoid, organelle genomes and virus genomes, as well as eukaryotic nuclear chromosomes, but only the last is considered in this chapter

* The original suggestion that genes were carried on chromosomes was based on the parallel behavior of chromosomes and Mendel ian genes (occurring in pairs, equal segregation, independent assortment). This is the chromosome theory of inheritance,

% There are three levels of duality in diploid eukaryotes. Chromosomes occur in homologous pairs, and when individual chromosomes become visible at mitosis, they can be seen to comprise a pair of chromatids, joined at a common centromere, each chromatid containing two DNA strands in a duplex. Despite the presence of eight DNA strands, the cells are still diploid. Only one strand of each DNA duplex actually carries the (transcribed) information, and the presence of two chromatids represents the doubling of information in preparation for segregation into daughter cells — cells are born with only one copy (a chromatid becomes a chromosome when it segregates). Furthermore, the paired chromatids of each mitotic chromosome are identical, whereas homologous chromosomes are not. Throughout the cell cycle, therefore, each locus is represented by just two alleles.

» Two types of chromosome can be distinguished at meiosis: autosomes, which have homo-morphic (structurally identical) partners and thus form homologous chromosome pairs, and heteiosomes (or allosomes) which have heteromorphic (structurally dissimilar) partners and form pairs which are homologous over only part of their lengths. The region of homology between two heteromorphic chromosomes is termed the pseudoautosomal region because the genes found within this region show the same pattern of inheritance as those situated on autosomes. Heterosomes are usually sex-chromosomes (q.v. pedigree analysis, sex-linked inheritance, sex determination).

*» The role of the chromosome is to provide a framework which allows each linear segment of the genome to replicate and segregate efficiently. Failure in either of those processes causes chromosome imbalance in daughter cells. Three specific as-acting sites are required for stable chromosome maintenance, an origin of replication (q.v,), a centromere and telomeres.

5.1 Normal chromosomes — gross morphology

Cytogenetic features of normal chromosomes. For most of the cell cycle, eukaryotic chromosomes exist as loosely packed chromatin and cannot be distinguished in the nucleus. They become visible at the onset of mitosis (or meiosis) when the chromatin condenses, forming discrete structures which stain densely when nuclei are treated with appropriate dyes (Bo* 5,1). The morphological features of the metaphase chromosome are shown in Figure 5.1, and these allow individual chromosomes to be recognized and aberrations to be identified (see Chromosome Mutation).

Chromosome banding. The features of chromosomes described in Figure 5,1 are based on homogeneous staining with DNA-binding dyes such as Feulgen. Mammalian chromosomes can aiso be stained in a heterogeneous manner using a variety of disruptive techniques which reveal a highly reproducible and specific pattern of alternate light and dark transverse bands (Table 5.1). Such chromosome banding methods allow chromosomes with similar gross morphology to be discriminated,

Chromosome <-rlrv the short ,'rm is designated p (pi-tite) and the |CAJ|

arm is designa lid q Satelil le Region (distallo

— the nucleolar urganizer region)

IVimary Constriction (centromere)

Secondary Constriction (nucleolar organiser region)

Figure 5.1: Morphological features of normal metaphase chromosomes. The primary constriction, which indicates the position of the centromere, stains densely and joins all four arms. Secondary constrictions are pale staining and usually represent nucleolar organizer regions (NORs), the positions of tandemly repeated rRNA genes, interstitial secondary constrictions are found on human chromosomes 1, 9 and 16, whilst distal constrictions appear on chromosomes 13, 14,15, 21 and 22. Telomeric chromosome segments found distal to NORs are termed satellite regions because they may appear detached from the main body of the chromosome. The satellite regions of different chromosomes are often found grouped together (satellite association), because the nucleolar organizers contribute to a common nucleolus. Other secondary constrictions can be induced by growing cells under abnormal conditions and are termed fragile sites (q.v.). Chromosome polymorphisms (heteromorphisms) are heritable morphological features of chromosomes which vary within a population but have no phenotype. Examples include areas of variable heterochromatin (as shown}, inversions (q.v.) and rare fragile sites. These can be used as cytogenetic markers.

Chromosome <-rlrv the short ,'rm is designated p (pi-tite) and the |CAJ|

arm is designa lid q Satelil le Region (distallo

— the nucleolar urganizer region)

IVimary Constriction (centromere)

Secondary Constriction (nucleolar organiser region)

Figure 5.1: Morphological features of normal metaphase chromosomes. The primary constriction, which indicates the position of the centromere, stains densely and joins all four arms. Secondary constrictions are pale staining and usually represent nucleolar organizer regions (NORs), the positions of tandemly repeated rRNA genes, interstitial secondary constrictions are found on human chromosomes 1, 9 and 16, whilst distal constrictions appear on chromosomes 13, 14,15, 21 and 22. Telomeric chromosome segments found distal to NORs are termed satellite regions because they may appear detached from the main body of the chromosome. The satellite regions of different chromosomes are often found grouped together (satellite association), because the nucleolar organizers contribute to a common nucleolus. Other secondary constrictions can be induced by growing cells under abnormal conditions and are termed fragile sites (q.v.). Chromosome polymorphisms (heteromorphisms) are heritable morphological features of chromosomes which vary within a population but have no phenotype. Examples include areas of variable heterochromatin (as shown}, inversions (q.v.) and rare fragile sites. These can be used as cytogenetic markers.

and provide a cytogenetic basis for gene mapping. They are most useful for characterizing chromosome aberrations. A low-resolution banding pattern is observed in metaphase chromosomes, but each metaphase band may be resolved into many sub-bands in the less condensed prometaphase chromosomes. The International System for Human Cytogenetic Nomenclature (ISCN1 is based on the bands and sub-bands in early and late prometaphase chromosomes and metaphase chromosomes (these have a resolution of 400, 550 and 850 bands, respectively). Each band is identified by chromosome and arm (e.g. lp, 2q) and is numbered from the centromere (ceil). Increasing resolution is designated by the use of more numbers (e.g. Ip3—>lp34—>lp34.1). Chromosome banding patterns reflect the structural and functional organization of the mammalian genome (q.v. isochores), with, for example, light Giemsa bands corresponding to regions of general transcriptional activity, early replication, low repetitive DNA content and DNase I sensitivity. This is only a very general organization, however, as each band corresponds to up to 10 Mb of DNA (see Chromatin, Genomes and Mapping). Lower eukaryotic chromosomes do not stain in response to the banding techniques applied to mammalian chromosomes, but the polytene chromosomes of dipteran insects display a natural banding pattern of high resolution (see below).

5.2 Special chromosome structures

A and B chromosomes. In most eukaryotes, each cell of a normal individual carries a defined and invariant set of chromosomes which are diagnostic of the species (q.v. karyotype; c.f. double minute chromosomes, gene amplification). Some species, however, carry extra chromosomes, often appearing to be composed entirely of heterochromatin, which have no effect on phenotype and often vary between populations, within a given population or even between cells within an individual. These structures, which are variously referred to as accessory chromosomes, satellite chromosomes, supernumerary chromosomes, or, in plants, B-chromosomes (to distinguish them from the invariant and essential A-chromosomes) often do not take part in mitosis and thus segregate randomly. They may be regarded as giant linear plasmids.

Polytene chromosomes. In Drosophila and other dipterans, the celts of certain larval secretory tissues (e.g. salivary glands) contain giant chromosomes comprising up to 1000 chromatids. These

Table 5.1: A selection of chromosome banding techniques

Banding technique

Method and applications

C-banding

Cj-banding D-banding

G-banding

G12-banding

N-bandirig Q-banding ft-banding

Replication banding

T-banding

A Giemsa-based technique which includes incubation with barium hydroxide. Identifies regions of heterochromatin (q.v.) and is therefore useful for determining the position of centromeres A technique for identifying kinetochores (q.v.) A technique for identifying regions of DNase I sensitivity, generally corresponding to regions of open chromatin which are potentially transcriptionally active The most widely applied chromosome banding technique, the reproducible pattern of bands being the basis of the international standard human and mouse cytogenetic maps. Chromosomes are partially digested with trypsin and incubated with Giemsa's stain (a mixture of methylene blue, eosin and other dyes dissolved in methanol) G-banding carried out at high pH, which stains mouse and human chromosomes differently, allowing them to be discriminated, e.g. in somatic cell hybrids

A technique for specifically staining nucleolar organizer regions, e.g. silver nitrate staining

A banding technique using the fluorescent dye quinacrine. This produces a similar pattern to G-banding, but also causes areas of heterochromatin to fluoresce brightly and is useful, e.g. for identifying the Y-chromosome A Giemsa-bassd technique including a heat-denatu rat ion step which results in a reversed banding pattern from that obtained with conventional G-banding. This technique is useful in, e.g. identifying terminal deletions This technique involves the incorporation of bramodeoxyuridine into replicating DNA. A brief pulse during the S phase allows replication timing to be investigated and if prolonged, generates a banding pattern similar to Giemsa's stain, suggesting G-bands correlate to replication time zones (q.v.). Incorporation over two rounds of replication allows sister chromatids to be discriminated on the basis that one has bramodeoxyuridine Incorporated In both strands and the other in only one strand. The chromatids then stain differently In the presence of Giemsa's stain and the fluorescent dye Hoechst 33258. This technique, known as harlequin staining, is useful for the detection of sister chromatid exchanges (q.v.) A variation of R-bandlng in which only telomeric DNA is stained. T-bands are particularly gene-rich [q.v. isochore, transcriptional mapping)

In many cases, these methods are empirical, i.e. it is not understood how they work. It is remarkable that these diverse techniques generate similar banding patterns, strongly indicating the structural organization of the genome into discrete isochores.

may also be termed poiytene chromosomes or Balbiani chromosomes after their discoverer, and are generated by multiple rounds of replication in the absence of mitosis as a strategy for gene amplification. Poiytene chromosomes are not only thicker than normal chromosomes, butare also longer because the association of many chromatids prevents the adoption of normal chromatin structure.

A remarkable feature of poiytene chromosomes is the highly reproducible pattern of transverse dark bands (or chromomeres) and light interbands. These are thought to reflect regional differences in chromatin density and to correspond functionally to individual chromatin domains (q.v.). The resolution of the banding pattern is much finer than even prometaphase bands on mammalian chromosomes (each poiytene band ranges in size from 1 to 100 kbp of DNA), but can be used in the same way, to construct detailed cytogenetic maps and characterize chromosome mutations.

A second feature of poiytene chromosomes is the presence of chromosome puffs and Balbiani rings. These are small and large, respectively, distensions of the chromosome which correspond to regions of transcriptional activity, presumably reflecting local decondensation of chromatin struc ture. Reproducible patterns of puffs and rings can be induced by certain environmental stimuli (e.g. treatment with the moulting hormone ecdysone), allowing the positions of the activated genes to be determined by direct observation.

Lampbrush chromosomes. In amphibian oocytes, where the cell cycle is arrested at diplonema (q.v.) of the first meiotic division, a single nucleus serves the needs of an extremely large cell. The four chromatids of the meiotic bivalent are held together by chiasmata, and can be seen to extrude long, uncoiled lateral loops of transcriptionally active chromatin from an axis of densely packed chro-momeres (the same term is used to describe the densely packed, presumed inactive, chromatin in both lampbrush and polytene chromosomes, and in decondensing mammalian chromosomes). The loops occur in pairs, one originating from each chromatid, and they are surrounded by a halo of ribonucleoprotein. This, and the extensively decondensed structure of the loops, is thought to reflect the continuous and prodigious transcriptional activity of the chromosome. There are some 10-15 000 loops active in the cell as a whole, but most of the DNA remains condensed as chromomeres. Like polytene bands and interbands, each loop is thought to correspond to a chromatin domain (q.v.).

5.3 Molecular aspects of chromosome structure

Molecular structures required for chromosome function. The gross morphology of the chromosome reveals little about how it functions in the cell. Primarily, the role of the chromosome is to provide a framework which allows each linear segment of the genome to replicate and segregate efficiently, failure in either of those processes resulting in chromosome imbalance in daughter cells (see Chromosome Mutation), Since each eukaryotic chromosome carries a different array of genes, the particular information carried on the chromosome does not influence its function. Three specific ris-acting sites are required for stable chromosome maintenance: an origin of replication (q.v.), a centromere and telomeres. The origin of replication is essential for the replication of the chromosome because it provides a site for the assembly of replication initiation proteins (see Replication). The centromere is essential for segregation because it provides a site for kinetochore assembly and facilitates microtubule attachment. The telomeres are essential for chromosome stability because they allow the completion of chromosome ends during DNA replication and prevent illegitimate end-joining to other chromosomes. Artificial chromosomes (q.v.) carrying arbitrary DNA can be stably co-maintained with the endogenous genome as long as these three elements are present.

Molecular nature of the centromere. In Saccharomyces cerevisiae, centromeres have been defined genetically by their ability to confer mitotic stability upon a plasmid (this is known as a CEN function). Several centromeres have been cloned by chromosome walking (q.v.) and appear to be functionally interchangeable. Sequence comparison has identified three conserved elements, termed CDI, CDII and CDII1. CDI has a short consensus sequence that appears to function primarily in meiotic segregation. CDII is an extremely AT-rich sequence of about 100 bp whose function is unclear. Mutations in both these elements influence mitotic segregation but do not abolish it, CDIII is a highly conserved 26 bp element displaying dyad symmetry, which appears to be essential for centromere activity, as point mutations at the centre of symmetry abolish centromere function, resulting in unstable segregation. A multiprotein complex termed Cbf-III binds to this element and displays microtubule motor activity. Cbf-III may thus represent the site of microtubule attachment and the engine for segregation at anaphase.

In Schizosaccharomyces pombe and higher eukaryotes, centromeres span several tens of kilobase pairs, compared with the minimal Saccharomyces cerevisiae centromere of 125 bp, and this may reflect the nature of spindle attachment. In S. cerevisiae, a single spindle fiber attaches to each chromosome, whereas in Schizosaccharomyces pombe and in mammals, numerous fibers are involved, and the centromere contains repetitive DNA. In S. pombe this displays dyad symmetry, whilst in mammals, the centromere contains a large proportion of satellite DNA (q.v.), which in primates consists of 170 bp tandem repeats with local perturbations. Proteins specifically associate with the mammalian centromere, one of which binds to satellite DNA in vitro and may represent the site of kinetochore formation.

Molecular nature of the telomere. The possession of a linear chromosome presents two problems to the eukaryotic cell. Firstly, because of the properties of DNj4 polymerases (q.v.), the 5' ends of each strand cannot be completed during replication {see Replication). Secondly, because of the abundance of end-joining enzymes for DNA repair, the chromosome ends could be ligated together generating polycentric compound chromosomes or ring chromosomes which would fail to segregate properly (see Chromosome Mutation). Both these potentially lethal processes are prevented by telomeres, which are specialized structures which are added to the chromosome ends in a replication-independent manner. Telomeres also appear to act as initiators of synapsis. They are associated with the nuclear envelope and are the first chromosome regions to pair up. In yeast and trypanosomes, subtelomeric DNA plays an important role in the regulation of gene expression by housing silent copies of information which is transferred to expression-competent sites by nonreciprocal recombination (q.v. mating type switching, antigen switching).

Telomeric DNA consists of short, tandemly repeated sequences. These have been characterized from a number of eukaryotes and are generally GC-rich, with guanidine residues clustered on one strand and cytidine residues on the other (Table 5.2). They may form unusual quadruplex structures by unorthodox interactions between guanosine residues, and which may play a role in protecting the telomere from end-joining reactions (see Nucleic Acid Structure). The addition of telomeric repeats to the termini of unstable linear plasmids confers stability as long as a centromere is also present.

Telomeres are added to the ends of DNA by a specialized ribonucleoprotein complex termed telomerase. This comprises several polypeptides and a single RNA molecule which contains two copies of the cytidine-rich strand sequence found in telomeric DNA. The protein component of telomerase possesses reverse transcriptase activity, but the activity appears to be limited to using the telomerase-specific RNA as a template. Based on this information, a current model suggests that telomere repeats are added to the 3' ends of existing telomeres by a primer extension/template translocation strategy, as shown in Figure 5.2. It is thought that the most distal telomere repeats can form a structure which blocks the telomere ends, and thus prevents illegitimate end-joining. This may involve looping of the DNA and/or the association with telomere-binding proteins. Looping of the terminal DNA could prime synthesis of the G-rich DNA strand.

Components of telomerase and other proteins associated with telomere activity can be identified from the analysis of mutations which affect telomere function. Mutations which affect telomere length, a strain-specific characteristic in yeast which is associated with a senescent phenotype, have identified the TLC1 gene (which encodes telomerase RNA) and several EST loci (even shorter telomeres) which code for telomerase polypeptides or telomere binding proteins like mammalian TRF1. The senescent phenotype suggests that telomeres may play a critical role in the life-span of a given cell. This is supported by the observation that the length of telomeres decreases with age in certain human somatic tissues, whilst it is maintained in germ cells (also q.v. growth transformation; see Oncogenes and Cancer). In mice, however, which have a much shorter life-span but much longer telomeres than humans, no age-dependent shortening has been observed. It has been suggested that

Table 5.2: Telomeric repeat sequences in different eukaryotes

Organism

Telomere repeat

Tetrahymena

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