JOURNALOF PATHOLOGY, VOL.
REVIEW ARTICLE-CHROMOSOME
163: 185-189 (1991)
PATHOLOGY
WHAT IS A CHROMOSOME? MORAG PARK
Department of Experimental Medicine, Ludwig Institute for Cancer Research, Montreal Branch, 687 Pine Avenue West, Hersey Pavillon, Montreal, Quebec, Canada H3A I A l
INTRODUCTION The term ‘chromosome’ (chroma, colour; soma, body) was used initially to describe the darkly staining bodies visible by light microscopy in eukaryotic cells at the time of cell division. Each chromosome contains a single DNA duplex and the total genetic information stored in the chromosomes of an organism constitutes its nuclear genome. The human diploid somatic cell nucleus contains 46 chromosomes with about 6 x lo9 nucleotide pairs of DNA. If represented as a continuous duplex, it would beapproximately 1.8 min length. An averagesized chromosome, therefore, contains about 40 mm of DNA packed into a nucleus 0.006 mm in diameter. To achieve this, the DNA duplex contained in each chromosome is condensed 6000-1 0 000-fold through several orders of DNA folding. Not all DNA is folded in the same way and the manner in which a region of the genome is packaged into chromatin will influence the activity of the genes contained within it. Understanding how the events of DNA metabolism, and DNA replication, recombination, and repair occur in DNA that is highly condensed into a chromatin structure and how this structure affects the selective expression of genes essential for cell differentiation and development is of obvious significance. Individual eukaryotic chromosomes are only microscopically visible during mitosis, when each sister chromatid pair consists of a fibre joined at the centromere (Fig. 1). During most of the cell cycle, when DNA is replicated and transcribed (interphase), individual chromosomes cannot be distinguished and the fibres are much less densely packed than in metaphase chromosomes. These states represent different degrees of condensation of the DNA such that DNA is organized in the chromosomes in a manner that allows cyclical states 0022-341 7/9 1/030185-05 $05.00 0 1991 by John Wiley & Sons, Ltd.
of condensation to exist between the packaging of chromatin at interphase and metaphase.
EUKARYOTIC CHROMOSOMES: MOLECULAR ORGANIZATION Nucleosome structure
The chromosome does not merely consist of DNA, but contains equal amounts by weight of basic histone proteins and acidic non-histone proteins. The complex of both classes of protein together with the nuclear DNA of eukaryotic cells forms chromatin. The histones play a crucial role in packing the DNA into its condensed structure. Histones are relatively small proteins with a high proportion of positively charged amino acids which assists the tight binding of histones with DNA regardless of the nucleotide sequence. The five types of histones fall into two groups: nucleosomal histones and H 1 histones. The nucleosomal histones form an octamer consisting of two copies of the H2A, H2B, H3, and H4 histones. Following digestion of chromatin with an enzyme that degrades DNA non-specifically,such as micrococcal nuclease, a portion of the DNA is protected from digestion and remains as double-stranded DNA fragments of 146 nucleotide pairs wound around (1.8 turns) the octamer histone core particle. Histone H1 is positioned outside the core structure, and is associated with the DNA as it enters and exits from its path around the inner histones. The proteins of the histone H1 class are polymorphic, are frequently modified during the cell cycle, and exhibit tissue and organism specificity. Nucleosomes are connected in tandem arrays by linker DNA which can vary in length from 0 to 80 bp. This structure can be visualized in electron micrographs as a series of
186
M. PARK DNA double helix
J
nucleosome particles plus linker DNA
11 nm
I
A
solenoid Chromatin fibre of packed nucleosomes
30 nm T
section of loops
] 300 nm
of chromatin fibres
telomere
condensed section of metaphase chromosome
+
metaphase chromosome
Fig. I-Folding
of DNA double helix into the metaphase chromosome
nucleosomes connected by a thread of free DNA (beads on a string, Fig. 1).
Nucleosome spacing Nucleosomes can be reconstituted in vitro irrespective of the DNA sequence. However, in vivo, nucleosomes do not appear to be distributed randomly on the DNA. Positioning of nucleosomes has been proposed as one mechanism by which the activity of the DNA is regulated. The regions of DNA from which nucleosomes are excluded may be created by the formation of DNA protein complexes that are concerned with controlling gene expression, DNA replication, or generating a higher-order chromatin structure.
When chromatin is examined in the electron microscope, two types of fibres are seen: a 10nm fibre composed of nucleosomes arranged edge to edge and a 30 nm fibre. The presence of histone H 1 is essential for winding nucleosornes into the 30 nm fibre, which contains a helical coil with approximately six nucleosomes for every turn. This condensed structure compresses the length of the DNA molecule roughly 3WO-fold. HIGHER-ORDER CHROMATIN STRUCTURE Chromatin domains Nucleosomes alone do not determine the higherorder folding of the chromatin fibre into
WHAT IS A CHROMOSOME?
chromosomes, where the ratio of compression is approximately 10 000-fold. Various models for this higher-order folding have been proposed, of which the loop model has substantial experimental support derived from electron microscopy, sedimentation, and nuclease digestion studies. In this model, the 30 nm fibre is folded into loops of 20-200 kilobase pairs that are held together at their bases by non-histone proteins (Fig. I). The role of histones in this model is to package the DNA of each loop, whereas other DNA binding proteins would organize the base of each loop. This model is supported by data showing that histone-depleted metaphase chromosomes still maintain a residual folded structure, with large DNA loops extending from a condensed proteinaceous lattice. These residual proteins form the chromosome scaffold of metaphase chromatin and act to constrain the DNA into looped domains. In condensed metaphase chromatin, such DNA loops are further coiled into a fibre of about 700 pm (Fig. 1) which is visible at the surface of intact metaphase chromosomes in transmission electron micrographs. In interphase cells this level ofchromatin organization persists. Chromatin is attached to a proteinaceous nuclear scaffold or matrix that extends throughout the nucleus and attaches to the cytoskeleton at the nuclear envelope. Several groups have identified DNA sequences that preferentially associate with either the metaphase scaffold [scaffold attachment regions (SARs)] or the interphase nuclear matrix, [matrix attachment regions (MARS)]. These sites are about 200 base pairs long, A-T rich, contain a consensus binding site for topoisomerase TI, are conserved in evolution, and often reside near cis-acting regulatory elements. The anchoring of DNA to the matrix may be necessary to allow matrix-bound enzymes and factors involved with DNA replication and transcription to interact with the DNA. This may be facilitated by the activity is a of topoisomerase 11. This enzyme-which quantitatively significant component of the nuclear matrix-has the ability to cleave both strands of the DNA double helix, pass a second double helix segment through the gap, and religate the original strands, a process which allows regions of the DNA to unwind. Structure of active chromatin Light and electron microscopy of interphase nuclei have for long suggested that chromatin exists in both condensed and decondensed forms (heterochromatin and euchromatin). It is not certain to
187
what extent these appearances of fixed structures accurately reflect chromatin configuration in life, but the relative sensitivity of the DNA to a nonspecific nuclease (DNasel) indicates that transcribed genes appear to be in an open decondensed state whereas non-transcribed genes are associated with condensed chromatin that is more resistant to cleavage. Active genes lie within large domains of chromatin, ranging in size from 10 to 100 kilobase pairs of DNA, which are preferentially sensitive to DNase1 compared with surrounding DNA. In addition, each domain possesses localized sites that are hypersensitive to DNase1 (DH sites). D H sites are short regions of DNA (50400 base pairs) that are frequently observed at or near the 5' end of genes. Many D H sites are believed to represent a region from which a nucleosome has been displaced by sequence-specific DNA binding proteins involved in gene regulation. Studies on cytologically amenable chromosomes such as the polytene chromosomes of Drosophilu melunoguster and lamprush chromosome loops in amphibian oocytes have revealed a relationship between chromatin loops and transcription units. The emerging model for DNA loop organization of transcribed genes proposes that flanking DNA sequences containing regulatory regions of genes or gene clusters are positioned close to the points where individual loops are attached to the nuclear matrix. The transcribed portion of the gene therefore lies within the loop. The hypothesis that a higher-order level of chromatin condensation is associated with a barrier to transcription is shown from certain types of genetic rearrangements that disrupt normal chromosomal boundaries of heterochromatin and lead to loss of gene expression associated with condensation of the adjoining euchromatin. Individual cloned genes introduced into the germ line of mice and Drosophila frequently show position effects on the level of the transferred gene expression. Similarly, regions of constitutive heterochromatin that are condensed in all cells are not transcribed. In humans, constitutive heterochromatin contains relatively simple repeated DNA sequences that form satellite DNAs which are predominantly localized around the centromere and at the telomeres. Other regions of DNA that can be condensed during interphase in some cell types of an organism and not in others form facultative heterochromatin, such as one of the X chromosomes in the somatic cells of female mammals. These regions are thought not to be transcribed following condensation.
188
M. PARK
Thus, genes can be packaged in a condensed form in which they may no longer be accessible to specific transcriptional-activator proteins. This supports the view that the compartmentalization of the genome into active and inactive domains during differentiation can exert an effect on the expression of genes placed near or within these domains.
Essential chromosomal elements In order for a DNA molecule to form a functional chromosome it must be able to replicate, segregate its two copies at mitosis, and maintain itself through cell generations. Studies on yeast chromosomes have identified the minimal DNA sequence elements required to maintain these chromosomal functions. In order to replicate, a DNA molecule requires a replication origin. Replication of the large amount of DNA contained in a eukaryotic chromosome is achieved by dividing it into many replicons. Replication origins have been precisely defined as particular DNA sequences in bacteria, viruses, and yeast. In humans, replication origins have not been well characterized but there is some evidence that they may be associated with MAR sites. It seems that the order in which replication origins are activated depends at least in part on the chromatin structure in which the origin is located, such that replicons in highly condensed chromatin replicate late and replicons in active or open chromatin replicate early. A second sequence element, the centromere, is involved in sister chromatid pairing prior to chromosome segregation and is the site of assembly for the kinetochore complex. The kinetochore is composed of a special subclass of chromatin fibres, which in human cells is composed of several million base pairs of A-T rich satellite DNA associated with specific non-histone proteins. The kinetochore complex mediates the attachment of the chromosome to the microtubules of the spindle apparatus, ensuring that each daughter cell will receive one of the newly replicated chromatids when the cell divides. The third essential element is the telomere, which forms the specialized end of a chromosome and serves to provide genetic stability to linear molecules. Free DNA ends are very recombinogenic and susceptible to exonucleotic attack. Without a telomere, DNA polymerases would be unable to replicate DNA at the ends of a chromosome; thus, it would become shorter with each cell generation. The telomeric DNA sequence is a simple G-rich repeat that can fold back to form a hairpin loop. A
specialized enzyme (telomerase) can add copies of this simple repeating sequence to the end of the chromosome, restoring the length of the chromosome. Telomeres may also provide positional information for chromosomes; in meiotic cells, they are attached to the nuclear membrane and are regions at which pairing of homologous chromosomes is initiated at mitosis. The domain model of chromosome organization provides an acceptable explanation for many cytological, biochemical, and genetic observations. This model can be used to explain the generation of some chromosome rearrangements, involving deletions, inversions, and translocations that result from non-homologous exchanges. Rearrangements occur most frequently in the period of the cell cycle during which DNA is replicated. It is proposed that rearrangements arise when a break in the DNA at a replication fork associated with one anchorage point is repaired by joining with DNA of a replication fork at a nearby anchorage point on the nuclear scaffold. The net effect of this breakage and reunion can result in deletion and inversion of the DNA between adjacent replication forks on the same chromosome or translocations when the DNA break is repaired with a replication fork on another chromosome. The enzymes involved in these reactions have not been well characterized in eukaryotes, although topoisomerase I1 has been shown to mediate illegitimate recombination in vitro. Why deletion-prone regions that contain no coding sequences and are associated with poor patient prognosis have not been lost in evolution could be explained if this region harbours indispensable functions such as sequences used in RNA processing or sites for chromosomal or nuclear matrix association. Continued research into the nature of chromatin structure and function should provide insight into how genes are regulated during development and how the chromosome structure has evolved. REFERENCES 1. Burch JBE, Weintraub H. Temporal order of chromatin structural
2. 3. 4. 5.
changes associated with activation of the major chicken vitellogenin gene. Cell 1983; 3 3 65-76. Eissenberg JC, Cartwright LL, Thomas GH, Elgin SCR. Selected topics in chromatin structure. Annu Rev Genet 1985; 1 9 485-536. Gasser SM, Laemnli UK. A glimpse at chromosomal order. Trends Genet 1987;3 1 6 2 2 . Gross DS, Garrard WT. Nuclease hypersensitive sites in chromatin. Annu RevBiochem 1988; 57: 159-198. Hastie ND, Allshire RC. Human telomeres: fusion and interstitial sites. Trends Genet 1989; 5: 326331.
W H A T IS A CHROMOSOME?
6. Nelson WG, Pienta KJ, Barrack ER, Coffey DS. The role of the nuclear matrix in the organization and function of DNA. Annu Rev Biophjs Biochem 1986: 15: 457475. 7. Rykowski MC, Parmelee SJ, Agard DA, Sedat JW. Precise determination of the molecular limits of a polytene chromosome band: regulatory sequences for the notch gene are in the interband. CeN 1988: W461-472. 8. Schimke RT, Sherwood SW, Hill AB, Johnston RN. Overreplication and recombination of D N A in higher eukaryotes: potential conse-
189
quences and biological implications. Proc N u f l Acud Sci USA 1986, 8 3 2157-2161. 9. Simpson RT. Nucleosome positioning can affect the function of a cis-acting D N A element in vivo. Nufure 1990: 343 387-390. 10. Vanin EF, Henthorn PS, Kioussis D, Grosveld F, Smithies 0. Unexpected relationships between four large deletions in the human 0-globin gene cluster. Cell 1983; 35: 701-709.
REVIEW ARTICLE-CHROMOSOME
163: 185-189 (1991)
PATHOLOGY
WHAT IS A CHROMOSOME? MORAG PARK
Department of Experimental Medicine, Ludwig Institute for Cancer Research, Montreal Branch, 687 Pine Avenue West, Hersey Pavillon, Montreal, Quebec, Canada H3A I A l
INTRODUCTION The term ‘chromosome’ (chroma, colour; soma, body) was used initially to describe the darkly staining bodies visible by light microscopy in eukaryotic cells at the time of cell division. Each chromosome contains a single DNA duplex and the total genetic information stored in the chromosomes of an organism constitutes its nuclear genome. The human diploid somatic cell nucleus contains 46 chromosomes with about 6 x lo9 nucleotide pairs of DNA. If represented as a continuous duplex, it would beapproximately 1.8 min length. An averagesized chromosome, therefore, contains about 40 mm of DNA packed into a nucleus 0.006 mm in diameter. To achieve this, the DNA duplex contained in each chromosome is condensed 6000-1 0 000-fold through several orders of DNA folding. Not all DNA is folded in the same way and the manner in which a region of the genome is packaged into chromatin will influence the activity of the genes contained within it. Understanding how the events of DNA metabolism, and DNA replication, recombination, and repair occur in DNA that is highly condensed into a chromatin structure and how this structure affects the selective expression of genes essential for cell differentiation and development is of obvious significance. Individual eukaryotic chromosomes are only microscopically visible during mitosis, when each sister chromatid pair consists of a fibre joined at the centromere (Fig. 1). During most of the cell cycle, when DNA is replicated and transcribed (interphase), individual chromosomes cannot be distinguished and the fibres are much less densely packed than in metaphase chromosomes. These states represent different degrees of condensation of the DNA such that DNA is organized in the chromosomes in a manner that allows cyclical states 0022-341 7/9 1/030185-05 $05.00 0 1991 by John Wiley & Sons, Ltd.
of condensation to exist between the packaging of chromatin at interphase and metaphase.
EUKARYOTIC CHROMOSOMES: MOLECULAR ORGANIZATION Nucleosome structure
The chromosome does not merely consist of DNA, but contains equal amounts by weight of basic histone proteins and acidic non-histone proteins. The complex of both classes of protein together with the nuclear DNA of eukaryotic cells forms chromatin. The histones play a crucial role in packing the DNA into its condensed structure. Histones are relatively small proteins with a high proportion of positively charged amino acids which assists the tight binding of histones with DNA regardless of the nucleotide sequence. The five types of histones fall into two groups: nucleosomal histones and H 1 histones. The nucleosomal histones form an octamer consisting of two copies of the H2A, H2B, H3, and H4 histones. Following digestion of chromatin with an enzyme that degrades DNA non-specifically,such as micrococcal nuclease, a portion of the DNA is protected from digestion and remains as double-stranded DNA fragments of 146 nucleotide pairs wound around (1.8 turns) the octamer histone core particle. Histone H1 is positioned outside the core structure, and is associated with the DNA as it enters and exits from its path around the inner histones. The proteins of the histone H1 class are polymorphic, are frequently modified during the cell cycle, and exhibit tissue and organism specificity. Nucleosomes are connected in tandem arrays by linker DNA which can vary in length from 0 to 80 bp. This structure can be visualized in electron micrographs as a series of
186
M. PARK DNA double helix
J
nucleosome particles plus linker DNA
11 nm
I
A
solenoid Chromatin fibre of packed nucleosomes
30 nm T
section of loops
] 300 nm
of chromatin fibres
telomere
condensed section of metaphase chromosome
+
metaphase chromosome
Fig. I-Folding
of DNA double helix into the metaphase chromosome
nucleosomes connected by a thread of free DNA (beads on a string, Fig. 1).
Nucleosome spacing Nucleosomes can be reconstituted in vitro irrespective of the DNA sequence. However, in vivo, nucleosomes do not appear to be distributed randomly on the DNA. Positioning of nucleosomes has been proposed as one mechanism by which the activity of the DNA is regulated. The regions of DNA from which nucleosomes are excluded may be created by the formation of DNA protein complexes that are concerned with controlling gene expression, DNA replication, or generating a higher-order chromatin structure.
When chromatin is examined in the electron microscope, two types of fibres are seen: a 10nm fibre composed of nucleosomes arranged edge to edge and a 30 nm fibre. The presence of histone H 1 is essential for winding nucleosornes into the 30 nm fibre, which contains a helical coil with approximately six nucleosomes for every turn. This condensed structure compresses the length of the DNA molecule roughly 3WO-fold. HIGHER-ORDER CHROMATIN STRUCTURE Chromatin domains Nucleosomes alone do not determine the higherorder folding of the chromatin fibre into
WHAT IS A CHROMOSOME?
chromosomes, where the ratio of compression is approximately 10 000-fold. Various models for this higher-order folding have been proposed, of which the loop model has substantial experimental support derived from electron microscopy, sedimentation, and nuclease digestion studies. In this model, the 30 nm fibre is folded into loops of 20-200 kilobase pairs that are held together at their bases by non-histone proteins (Fig. I). The role of histones in this model is to package the DNA of each loop, whereas other DNA binding proteins would organize the base of each loop. This model is supported by data showing that histone-depleted metaphase chromosomes still maintain a residual folded structure, with large DNA loops extending from a condensed proteinaceous lattice. These residual proteins form the chromosome scaffold of metaphase chromatin and act to constrain the DNA into looped domains. In condensed metaphase chromatin, such DNA loops are further coiled into a fibre of about 700 pm (Fig. 1) which is visible at the surface of intact metaphase chromosomes in transmission electron micrographs. In interphase cells this level ofchromatin organization persists. Chromatin is attached to a proteinaceous nuclear scaffold or matrix that extends throughout the nucleus and attaches to the cytoskeleton at the nuclear envelope. Several groups have identified DNA sequences that preferentially associate with either the metaphase scaffold [scaffold attachment regions (SARs)] or the interphase nuclear matrix, [matrix attachment regions (MARS)]. These sites are about 200 base pairs long, A-T rich, contain a consensus binding site for topoisomerase TI, are conserved in evolution, and often reside near cis-acting regulatory elements. The anchoring of DNA to the matrix may be necessary to allow matrix-bound enzymes and factors involved with DNA replication and transcription to interact with the DNA. This may be facilitated by the activity is a of topoisomerase 11. This enzyme-which quantitatively significant component of the nuclear matrix-has the ability to cleave both strands of the DNA double helix, pass a second double helix segment through the gap, and religate the original strands, a process which allows regions of the DNA to unwind. Structure of active chromatin Light and electron microscopy of interphase nuclei have for long suggested that chromatin exists in both condensed and decondensed forms (heterochromatin and euchromatin). It is not certain to
187
what extent these appearances of fixed structures accurately reflect chromatin configuration in life, but the relative sensitivity of the DNA to a nonspecific nuclease (DNasel) indicates that transcribed genes appear to be in an open decondensed state whereas non-transcribed genes are associated with condensed chromatin that is more resistant to cleavage. Active genes lie within large domains of chromatin, ranging in size from 10 to 100 kilobase pairs of DNA, which are preferentially sensitive to DNase1 compared with surrounding DNA. In addition, each domain possesses localized sites that are hypersensitive to DNase1 (DH sites). D H sites are short regions of DNA (50400 base pairs) that are frequently observed at or near the 5' end of genes. Many D H sites are believed to represent a region from which a nucleosome has been displaced by sequence-specific DNA binding proteins involved in gene regulation. Studies on cytologically amenable chromosomes such as the polytene chromosomes of Drosophilu melunoguster and lamprush chromosome loops in amphibian oocytes have revealed a relationship between chromatin loops and transcription units. The emerging model for DNA loop organization of transcribed genes proposes that flanking DNA sequences containing regulatory regions of genes or gene clusters are positioned close to the points where individual loops are attached to the nuclear matrix. The transcribed portion of the gene therefore lies within the loop. The hypothesis that a higher-order level of chromatin condensation is associated with a barrier to transcription is shown from certain types of genetic rearrangements that disrupt normal chromosomal boundaries of heterochromatin and lead to loss of gene expression associated with condensation of the adjoining euchromatin. Individual cloned genes introduced into the germ line of mice and Drosophila frequently show position effects on the level of the transferred gene expression. Similarly, regions of constitutive heterochromatin that are condensed in all cells are not transcribed. In humans, constitutive heterochromatin contains relatively simple repeated DNA sequences that form satellite DNAs which are predominantly localized around the centromere and at the telomeres. Other regions of DNA that can be condensed during interphase in some cell types of an organism and not in others form facultative heterochromatin, such as one of the X chromosomes in the somatic cells of female mammals. These regions are thought not to be transcribed following condensation.
188
M. PARK
Thus, genes can be packaged in a condensed form in which they may no longer be accessible to specific transcriptional-activator proteins. This supports the view that the compartmentalization of the genome into active and inactive domains during differentiation can exert an effect on the expression of genes placed near or within these domains.
Essential chromosomal elements In order for a DNA molecule to form a functional chromosome it must be able to replicate, segregate its two copies at mitosis, and maintain itself through cell generations. Studies on yeast chromosomes have identified the minimal DNA sequence elements required to maintain these chromosomal functions. In order to replicate, a DNA molecule requires a replication origin. Replication of the large amount of DNA contained in a eukaryotic chromosome is achieved by dividing it into many replicons. Replication origins have been precisely defined as particular DNA sequences in bacteria, viruses, and yeast. In humans, replication origins have not been well characterized but there is some evidence that they may be associated with MAR sites. It seems that the order in which replication origins are activated depends at least in part on the chromatin structure in which the origin is located, such that replicons in highly condensed chromatin replicate late and replicons in active or open chromatin replicate early. A second sequence element, the centromere, is involved in sister chromatid pairing prior to chromosome segregation and is the site of assembly for the kinetochore complex. The kinetochore is composed of a special subclass of chromatin fibres, which in human cells is composed of several million base pairs of A-T rich satellite DNA associated with specific non-histone proteins. The kinetochore complex mediates the attachment of the chromosome to the microtubules of the spindle apparatus, ensuring that each daughter cell will receive one of the newly replicated chromatids when the cell divides. The third essential element is the telomere, which forms the specialized end of a chromosome and serves to provide genetic stability to linear molecules. Free DNA ends are very recombinogenic and susceptible to exonucleotic attack. Without a telomere, DNA polymerases would be unable to replicate DNA at the ends of a chromosome; thus, it would become shorter with each cell generation. The telomeric DNA sequence is a simple G-rich repeat that can fold back to form a hairpin loop. A
specialized enzyme (telomerase) can add copies of this simple repeating sequence to the end of the chromosome, restoring the length of the chromosome. Telomeres may also provide positional information for chromosomes; in meiotic cells, they are attached to the nuclear membrane and are regions at which pairing of homologous chromosomes is initiated at mitosis. The domain model of chromosome organization provides an acceptable explanation for many cytological, biochemical, and genetic observations. This model can be used to explain the generation of some chromosome rearrangements, involving deletions, inversions, and translocations that result from non-homologous exchanges. Rearrangements occur most frequently in the period of the cell cycle during which DNA is replicated. It is proposed that rearrangements arise when a break in the DNA at a replication fork associated with one anchorage point is repaired by joining with DNA of a replication fork at a nearby anchorage point on the nuclear scaffold. The net effect of this breakage and reunion can result in deletion and inversion of the DNA between adjacent replication forks on the same chromosome or translocations when the DNA break is repaired with a replication fork on another chromosome. The enzymes involved in these reactions have not been well characterized in eukaryotes, although topoisomerase I1 has been shown to mediate illegitimate recombination in vitro. Why deletion-prone regions that contain no coding sequences and are associated with poor patient prognosis have not been lost in evolution could be explained if this region harbours indispensable functions such as sequences used in RNA processing or sites for chromosomal or nuclear matrix association. Continued research into the nature of chromatin structure and function should provide insight into how genes are regulated during development and how the chromosome structure has evolved. REFERENCES 1. Burch JBE, Weintraub H. Temporal order of chromatin structural
2. 3. 4. 5.
changes associated with activation of the major chicken vitellogenin gene. Cell 1983; 3 3 65-76. Eissenberg JC, Cartwright LL, Thomas GH, Elgin SCR. Selected topics in chromatin structure. Annu Rev Genet 1985; 1 9 485-536. Gasser SM, Laemnli UK. A glimpse at chromosomal order. Trends Genet 1987;3 1 6 2 2 . Gross DS, Garrard WT. Nuclease hypersensitive sites in chromatin. Annu RevBiochem 1988; 57: 159-198. Hastie ND, Allshire RC. Human telomeres: fusion and interstitial sites. Trends Genet 1989; 5: 326331.
W H A T IS A CHROMOSOME?
6. Nelson WG, Pienta KJ, Barrack ER, Coffey DS. The role of the nuclear matrix in the organization and function of DNA. Annu Rev Biophjs Biochem 1986: 15: 457475. 7. Rykowski MC, Parmelee SJ, Agard DA, Sedat JW. Precise determination of the molecular limits of a polytene chromosome band: regulatory sequences for the notch gene are in the interband. CeN 1988: W461-472. 8. Schimke RT, Sherwood SW, Hill AB, Johnston RN. Overreplication and recombination of D N A in higher eukaryotes: potential conse-
189
quences and biological implications. Proc N u f l Acud Sci USA 1986, 8 3 2157-2161. 9. Simpson RT. Nucleosome positioning can affect the function of a cis-acting D N A element in vivo. Nufure 1990: 343 387-390. 10. Vanin EF, Henthorn PS, Kioussis D, Grosveld F, Smithies 0. Unexpected relationships between four large deletions in the human 0-globin gene cluster. Cell 1983; 35: 701-709.
