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Lampbrush Chromosomes
L a m p b r u s h C h ro m o s o m e s 1073 science: the Russian agronomist Trofim Lysenko, who dominated (not to say destroyed) genetics in the Soviet Union and its satellite popular democracies from the mid-1930s until the mid-1960s. Lysenko was not openly opposed to classical Darwinism, Karl Marx having been a great admirer of Darwin but he declared neo-Darwinism, with its reliance on Mendelian genetics and gene mutation, to be idealist± racist metaphysical speculations propagated by the Catholic Church and the Fascists to keep the proletariat intellectually enchained. At first, in the 1930s, Lysenko denied that he was a Lamarckist and declared that ``starting from Lamarckian positions, the work of remaking the nature of plants by `education' cannot lead to positive results.'' Then, when he became director of the Institute of Genetics of the Soviet Academy of Sciences in 1940 and had Stalin's ear, Lysenko declared Mendelian genetics erroneous. By 1948, when he had ruthlessly silenced any Soviet geneticists who opposed him, he no longer concealed his adherence to Lamarckism, declaring that: the well-known Lamarckian propositions, which recognize the active role of the conditions of the external environment in the living body and the inheritance of acquired characters, in contrast to the metaphysics of neo-Darwinism, are indeed scientific.
Lysenko was finally dismissed in 1965, after having gravely hampered scientific and agricultural progress in the Soviet Union for more than 25 years. Nevertheless, `Lamarckist' remains a term of ridicule. This is a most regrettable affront to the memory of one of the great figures in the history of biology, to whom that discipline owes its very name. See also: Lamarck, Jean Baptiste; Lysenko, T.D./ Lysenkoism
Lampbrush Chromosomes
lampbrushy, and then contract again to form normal first meiotic metaphase bivalents. They are characterized by widespread RNA transcription from hundreds of transcription units that are arranged at short intervals along the lengths of all the chromosomes. LBCs were first seen in salamander oocytes by Flemming in 1882 and in oocytes of a dogfish by Ruckert in 1892. The name lampbrush originated from Ruckert, who likened the objects to a nineteenthcentury lampbrush, equivalent to the modern testtube brush. LBCs are delicate structures and they must be carefully dissected out of their nuclei in order to examine them in a life-like condition. The largest LBCs are to be found in oocytes of newts and salamanders, animals that have large genomes and correspondingly large LBCs. The best oocytes for lampbrush studies are those that make up the bulk of the ovary of a healthy adult female at the time of year when the eggs are actively growing. They are about 1 mm in diameter and their nuclei are between 0.3 and 0.5 mm in diameter (Figure 1). The techniques for isolating and looking at LBCs from such oocytes are specialized but inexpensive and simple; details are available in the sources cited in the Further Reading section. Since an LBC is a meiotic half bivalent, it must consist of two chromatids. The entire lampbrush bivalent will therefore have a total of four chromatids. The chromosome appears as a row of granules of deoxyribonucleoprotein (DNP), the chromomeres, connected by an exceedingly thin thread of the same material (Figure 2). Chromomeres are 0.25±2 mm in diameter and are spaced 1±2 mm center to center along the chromosome. Each chromomere has two or a multiple of two loops associated with it. The loops have a thin axis of DNP surrounded by a loose matrix of ribonucleoprotein (RNP). The loops are variable in length, ranging from about 5 to 100 mm. Loops vary in appearance. Loops of the same appearance always occur at the same locus on the same chromosome within a species.
H C Macgregor Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.0745
Lampbrush chromosomes (LBCs) are elongated diplotene bivalents in prophase of the first meiotic division in growing oocytes in the ovaries of most animals other than mammals and certain insects. Some LBCs reach lengths of a millimeter or more. The chromosomes go from a compact telophase form at the end of the last oogonial mitosis, become
Nuclear membrane Nucleus − containing lampbrush chromosomes Cytoplasm and yolk
Figure 1 An oocyte (growing ovarian egg) showing the relative dimensions of the egg, its nucleus, and its lampbrush chromosome.
1074
L a m p b r u s h C h ro m o s o m e s P PPP
L
LL
C cf
Figure 2 A region of a lampbrush chromosome showing the interchromomeric axial fiber (cf ) connecting small compact chromomeres (c), chromomeres bearing pairs (L) or multiple pairs (LL) of loops, loops of different morphologies, polarization of thickness along individual loops, loops consisting of a single unit of polarization (P), and loops with several tandem units of polarization having the same or different directions of polarity (PPP). Some particularly distinctive loops can be used for chromosome identification and the construction of LBC maps. Loops arising from the same chromomere have the same appearance and are usually, though not always, of the same length (Figure 2). The general pattern of events during the lampbrush phase of oogenesis is one of extension followed by retraction of the lampbrush loops and there is a clear inverse relationship between loop length and chromomere size. The longer the loop, the smaller the chromomere, and vice versa. Most lateral loops have an asymmetrical form. They are thin at one end of insertion into their chromomere and become progressively thicker towards the other end (Figure 2). If an LBC is stretched, breaks first happen transversely across the chromomeres so that the resulting gaps are spanned by the loops that are associated with the chromomeres (Figure 3). This demonstrates the structural continuity between the main axis of the chromosome ± the interchromomeric fiber ± and the axes of the loops. Lampbrush loops are sites of active RNA synthesis and RNA is being transcribed simultaneously all along the length of the loop. In newts, there are more than 20 000 RNA-synthesizing loops per oocyte. Particular loops may be present or absent in homozygous or heterozygous combinations and the frequency of combinations within and between bivalents with respect to presence or absence of loops, signifies that these loops assort and recombine like pairs of Mendelian alleles. So there appears to be an element of genetic unity in a loop±chromomere complex. By 1960, it was known that an LBC has two DNA duplexes running alongside one another in the interchromomeric fiber, compacted into chromomeres at intervals and extending laterally from a point within
Figure 3 Breakage of a stretched lampbrush chromosome across a chromomere, such that loops associated with the chromomeres come to span the gap between the two halves of the chromomere. each chromomere to form loops where RNA transcription takes place. Each duplex represents one chromatid (Figure 4). New technologies of the late 1970s confirmed this model and extended it. A technique that removed most of the protein from chromosomes, leaving only the DNA and attached newly transcribed RNA, and then visualized what was left by electron microscopy, showed a lampbrush loop as a thin DNA axis with RNA polymerase molecules lined up and closely packed along its entire length. Each polymerase carried a strand of RNP. At one end of the DNA axis, the RNP strands are short.
L a m p b r u s h C h ro m o s o m e s 1075
Figure 4 Accepted model of lampbrush chromosome organization showing the interchromomeric fiber consisting of two chromatids that separate from one another and become involved in RNA transcription in the regions of the loops. At the other end, they are much longer and they show a smooth gradient in size from one end to the other. In essence, the entire region is polarized and asymmetric, in the same sense as a loop, as seen with the light microscope, is asymmetric. The DNA axis outside the region occupied by polymerases shows the structure that would be expected of nontranscribing chromatin. The lengths of the transcribed regions of the chromosome are about the same as the lengths of loops as seen and measured with light microscopy. Lampbrush loops are therefore polarized units of transcription. The polymerase moves on a stationary loop axis. A loop is formed by an initial `spinning out' process, probably powered by the continuing attachment of more and more polymerases to a specific region of the chromomeric DNA. The loop remains and is transcribed as a permanent structure throughout the lampbrush phase. Towards the end of the lampbrush phase, transcriptive activity declines, polymerases detach from loop axes, and loops regress and disappear. The vast majority of the chromomeric DNA is never transcribed and a loop represents a short, specific part of the DNA in a loop±chromomere complex. In situ nucleic acid hybridization is a means of locating specific gene sequences on chromosomes. Let us suppose that each loop represents `a gene.' The RNA that makes up the loop matrix, the attached nascent transcripts, will all be or include transcripts of
that `gene.' In effect, the loop is a large object, consisting of hundreds of RNA copies of the gene, all clustered at one position on the chromosome set. Isolate and purify the DNA of that gene, and label it in some way, and it will be easy to make it single-stranded and bind it specifically to the complementary singlestranded RNA attached to the lampbrush loop. The technique is known as DNA/RNA transcript in situ hybridization (DR/ISH). The end product of an experiment involving DR/ ISH is a preparation showing one or more pairs of loops with label distributed along their lengths. It is not uncommon in DR/ISH experiments to find loops that are labeled over only part of their lengths. This is evidence that the DNA sequence of a loop axis can and does change from place to place along the length of the loop. Wherever there are partially labeled loops, it is usual to find the same partially labeled loops, with precisely the same pattern of labeling, in every oocyte over quite a wide range of size and stage. So loops are permanent structures that transcribe from the same stretch of DNA axis throughout the entire lampbrush phase. DR/ISH experiments prove that highly repeated short DNA sequences, commonly referred to as `satellite' DNA, which could not possibly serve as a basis for transcription and translation into functional polypeptides, are abundantly transcribed on lampbrush loops along with more complex sequences that are definitely translated into functional proteins. The current hypothesis for LBC function is as follows. At the thin base of each loop or the start of each transcription unit there is a promoter site for a functional gene sequence. RNA polymerase attaches to this site and moves along the DNA, transcribing the sense strand of the gene and generating messenger RNA molecules that remain attached to the polymerase (Figure 5). In the lampbrush environment there are no stop signals for transcription, so the polymerases continue to transcribe past the end of the functional gene and into whatever DNA sequences lie `downstream' of the gene. This results in very long transcription units, very long transcripts, mixing of gene transcripts with nonsense transcripts in high molecular weight nuclear RNA, and lampbrush loops. This `read-through' hypothesis predicts that the number of functional genes that are expressed to form translatable RNAs may be expected to equal the number of transcription units that are active in a lampbrush set. The hypothesis says, in effect, that the only unusual feature of an LBC, and the very reason for the lampbrush form, is that once transcription starts it cannot stop until the polymerase meets another promoter that is already initiated or some condensed chromomeric chromatin that is physically impenetrable and untranscribable.
1076
L a m p b r u s h C h ro m o s o m e s
Figure 5 Transcription on a lampbrush chromosome loop where a gene (thick black line) is transcribed from its promoter (black flag) through to and past its normal stop signal (white flag) and into the normally nontranscribed DNA that lies downstream, thus generating very long transcription units with long transcripts that include RNA complementary to the sense strand of the gene (thick parts of the transcripts) and nonsense DNA that lies downstream of the gene (thin parts of the transcripts).
Evidence for the Read-Through Hypothesis for LBCs LBCs dissected directly into a solution of the enzyme deoxyribonuclease-1 (DNase-1) fall to pieces and their loops break into thousands of fragments. This does not happen with ribonuclease or proteases. If breakage of the chromosome axis and the loops by DNase is watched and timed and the number of breaks plotted against time on a log scale, the slope of the plot for the chromosome axis is 4 and that for the loops is 2. This supports the model in which the axis consists of two chromatids ± each a DNA double helix consisting of two nucleotide chains ± and the loop is part of one chromatid ± consisting of one double helix made up from two nucleotide chains. A later experiment used restriction enzymes that cleaved DNA only at places along the molecule where there was a particular short nucleotide sequence. If a loop consisted entirely of identical tandemly repeated DNA sequences, all with a particular restriction
enzyme recognition site, then the loop would be destroyed by that enzyme. If, on the other hand, the DNA sequences all lacked the enzyme recognition site, then the loop would be totally unaffected and would remain intact. An experiment was set up using five enzymes and the LBCs from N. viridescens. The control enzyme was deoxyribonuclease-1. DNase-1 and three of the restriction enzymes destroyed everything. One enzyme, HaeIII did likewise, except that it left one pair of loops completely intact. These HaeIII resistant loops were big ones, 100 mm long, equivalent to at least 300 000 nucleotides. Their unique resistance to HaeIII provided direct evidence that at least one pair of loops consisted of tandemly repeated short sequence DNA. At a later date, the effects of HaeIII were tested again, with appropriate controls, on the HaeIII resistant loops of N. viridescens. Breaks regularly occurred precisely at the thin beginnings of each loop, but the remainder of the loops remained intact, as would be predicted on the basis of the read-through hypothesis. The start of the transcription unit would be characterized by a long complex gene sequence that would almost inevitably include the HaeIII recognition site. The remainder of the loop would consist entirely of repeat sequences that lacked the HaeIII site.
Other Questions We Should Ask about Lampbrushes Only a small fraction of the entire DNA of a loop± chromomere complex forms the transcription unit that makes the loop. What about the rest of the DNA? Is the DNA segment that makes the loop preferentially selected for transcription the same piece at the corresponding locus in every egg of every individual of a particular species? This question may be approached experimentally. Why do loops have different morphologies that are heritable, locus-specific, and sometimes speciesspecific? The loop matrix is a site of processing, cleaving, and packaging of nuclear RNA, so most of the variation in gross structure may be expected to reflect different modes of binding and interaction involving quite a wide range of proteins and RNAs. Do LBCs look the same in all animals? They do not. The relative lengths of LBCs at the time of their maximum development are the same as the relative lengths of the corresponding mitotic metaphase chromosomes from the same species. The overall lengths of LBC are broadly related to genome size. Birds, with their notably small genomes, have extremely small, but nonetheless very beautiful, LBC that present many extraordinary and hitherto unexplained features.
L e a d er Pe p t i d e 1077 Some LBCs have long loops and others have very short ones. We have seen that the transcription units of LBCs are unusually long because they include interspersed repetitive elements of the genome. Structural genes in large genomes are more widely spaced than in small genomes as they are interspersed with noncoding DNA. One might therefore expect LBCs from large genomes to have longer loops (transcription units) than those of smaller genomes, and this is what has been observed. Many of the very long loops that we see in LBC from animals with large genomes show multiple, tandemly arranged thin±thick segments (transcription units). The individual transcription units within one loop can have the same or opposite polarities and can be of the same or different lengths (Figure 6). This observation suggests that it is really the transcription unit that is the ultimate genetic unit in an LBC and not the loop/chromomere complex, as was once thought. Why do LBCs exist at all? They are characteristic of eggs that develop quickly into complex multicellular organisms independently of the parent. A frog's egg is fertilized and develops into a complex tadpole within a few days. Much of the information and raw materials for this process are laid down during oogenesis through activity of LBCs and amplified ribosomal genes and the accumulation of yolk proteins imported from the liver. LBCs may therefore be regarded as an adaptive feature that has evolved to preprogramme the egg for rapid early development. The fact that they are not present in mammalian eggs could be regarded as an advanced feature that is consistent with the slow pace of mammalian development. A frog's egg, for example, will have completed gastrulation and the differentiation of its central nervous system and
embryonic axis by the time a human embryo has only reached the 8-cell stage. LBCs provide a uniquely powerful medium through which it has been possible to draw valid conclusions at the molecular level from observations and experiments carried out mainly with a light microscope. Their value extends into the fields of comparative molecular cytogenetics and systematics. Nowhere else is it possible to study genome structure, function, and diversity by actually looking at the genome itself with a light microscope. LBCs are technically challenging but not defeating. They are exceptionally beautiful to look at and fun to work with. Further information on these remarkable structures can be found in the literature listed in the Further Reading section below and on the internet.
Further Reading
Callan G (1986) Lampbrush Chromosomes. Berlin: SpringerVerlag. Macgregor HC (1993) An Introduction to Animal Cytogenetics. London: Chapman & Hall. Macgregor HC and Varley J (1988) Working with Animal Chromosomes. New York: John Wiley.
See also: Cytogenetics; Developmental Genetics
Late Genes E Kutter Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.0746
During viral infection, late genes are those that are transcribed after the commencement of viral DNA synthesis. The bulk of these encode either components of the capsid, proteins aiding in morphogenesis or DNA packaging, or proteins that are to be carried with the DNA in the capsid. See also: Virus
Leader Peptide Figure 6 The various arrangements of transcription units that actually occur on lampbrush chromosomes. The loop on the left comprises a single transcription unit. In the middle loop there are two transcription units of the same size and polarity. The right-hand loop has four transcription units of different sizes and different directions of polarity.
J Parker Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.0749
The term `leader peptide' (or, less commonly, `leader polypeptide') refers to a peptide encoded by a DNA
Lysenko was finally dismissed in 1965, after having gravely hampered scientific and agricultural progress in the Soviet Union for more than 25 years. Nevertheless, `Lamarckist' remains a term of ridicule. This is a most regrettable affront to the memory of one of the great figures in the history of biology, to whom that discipline owes its very name. See also: Lamarck, Jean Baptiste; Lysenko, T.D./ Lysenkoism
Lampbrush Chromosomes
lampbrushy, and then contract again to form normal first meiotic metaphase bivalents. They are characterized by widespread RNA transcription from hundreds of transcription units that are arranged at short intervals along the lengths of all the chromosomes. LBCs were first seen in salamander oocytes by Flemming in 1882 and in oocytes of a dogfish by Ruckert in 1892. The name lampbrush originated from Ruckert, who likened the objects to a nineteenthcentury lampbrush, equivalent to the modern testtube brush. LBCs are delicate structures and they must be carefully dissected out of their nuclei in order to examine them in a life-like condition. The largest LBCs are to be found in oocytes of newts and salamanders, animals that have large genomes and correspondingly large LBCs. The best oocytes for lampbrush studies are those that make up the bulk of the ovary of a healthy adult female at the time of year when the eggs are actively growing. They are about 1 mm in diameter and their nuclei are between 0.3 and 0.5 mm in diameter (Figure 1). The techniques for isolating and looking at LBCs from such oocytes are specialized but inexpensive and simple; details are available in the sources cited in the Further Reading section. Since an LBC is a meiotic half bivalent, it must consist of two chromatids. The entire lampbrush bivalent will therefore have a total of four chromatids. The chromosome appears as a row of granules of deoxyribonucleoprotein (DNP), the chromomeres, connected by an exceedingly thin thread of the same material (Figure 2). Chromomeres are 0.25±2 mm in diameter and are spaced 1±2 mm center to center along the chromosome. Each chromomere has two or a multiple of two loops associated with it. The loops have a thin axis of DNP surrounded by a loose matrix of ribonucleoprotein (RNP). The loops are variable in length, ranging from about 5 to 100 mm. Loops vary in appearance. Loops of the same appearance always occur at the same locus on the same chromosome within a species.
H C Macgregor Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.0745
Lampbrush chromosomes (LBCs) are elongated diplotene bivalents in prophase of the first meiotic division in growing oocytes in the ovaries of most animals other than mammals and certain insects. Some LBCs reach lengths of a millimeter or more. The chromosomes go from a compact telophase form at the end of the last oogonial mitosis, become
Nuclear membrane Nucleus − containing lampbrush chromosomes Cytoplasm and yolk
Figure 1 An oocyte (growing ovarian egg) showing the relative dimensions of the egg, its nucleus, and its lampbrush chromosome.
1074
L a m p b r u s h C h ro m o s o m e s P PPP
L
LL
C cf
Figure 2 A region of a lampbrush chromosome showing the interchromomeric axial fiber (cf ) connecting small compact chromomeres (c), chromomeres bearing pairs (L) or multiple pairs (LL) of loops, loops of different morphologies, polarization of thickness along individual loops, loops consisting of a single unit of polarization (P), and loops with several tandem units of polarization having the same or different directions of polarity (PPP). Some particularly distinctive loops can be used for chromosome identification and the construction of LBC maps. Loops arising from the same chromomere have the same appearance and are usually, though not always, of the same length (Figure 2). The general pattern of events during the lampbrush phase of oogenesis is one of extension followed by retraction of the lampbrush loops and there is a clear inverse relationship between loop length and chromomere size. The longer the loop, the smaller the chromomere, and vice versa. Most lateral loops have an asymmetrical form. They are thin at one end of insertion into their chromomere and become progressively thicker towards the other end (Figure 2). If an LBC is stretched, breaks first happen transversely across the chromomeres so that the resulting gaps are spanned by the loops that are associated with the chromomeres (Figure 3). This demonstrates the structural continuity between the main axis of the chromosome ± the interchromomeric fiber ± and the axes of the loops. Lampbrush loops are sites of active RNA synthesis and RNA is being transcribed simultaneously all along the length of the loop. In newts, there are more than 20 000 RNA-synthesizing loops per oocyte. Particular loops may be present or absent in homozygous or heterozygous combinations and the frequency of combinations within and between bivalents with respect to presence or absence of loops, signifies that these loops assort and recombine like pairs of Mendelian alleles. So there appears to be an element of genetic unity in a loop±chromomere complex. By 1960, it was known that an LBC has two DNA duplexes running alongside one another in the interchromomeric fiber, compacted into chromomeres at intervals and extending laterally from a point within
Figure 3 Breakage of a stretched lampbrush chromosome across a chromomere, such that loops associated with the chromomeres come to span the gap between the two halves of the chromomere. each chromomere to form loops where RNA transcription takes place. Each duplex represents one chromatid (Figure 4). New technologies of the late 1970s confirmed this model and extended it. A technique that removed most of the protein from chromosomes, leaving only the DNA and attached newly transcribed RNA, and then visualized what was left by electron microscopy, showed a lampbrush loop as a thin DNA axis with RNA polymerase molecules lined up and closely packed along its entire length. Each polymerase carried a strand of RNP. At one end of the DNA axis, the RNP strands are short.
L a m p b r u s h C h ro m o s o m e s 1075
Figure 4 Accepted model of lampbrush chromosome organization showing the interchromomeric fiber consisting of two chromatids that separate from one another and become involved in RNA transcription in the regions of the loops. At the other end, they are much longer and they show a smooth gradient in size from one end to the other. In essence, the entire region is polarized and asymmetric, in the same sense as a loop, as seen with the light microscope, is asymmetric. The DNA axis outside the region occupied by polymerases shows the structure that would be expected of nontranscribing chromatin. The lengths of the transcribed regions of the chromosome are about the same as the lengths of loops as seen and measured with light microscopy. Lampbrush loops are therefore polarized units of transcription. The polymerase moves on a stationary loop axis. A loop is formed by an initial `spinning out' process, probably powered by the continuing attachment of more and more polymerases to a specific region of the chromomeric DNA. The loop remains and is transcribed as a permanent structure throughout the lampbrush phase. Towards the end of the lampbrush phase, transcriptive activity declines, polymerases detach from loop axes, and loops regress and disappear. The vast majority of the chromomeric DNA is never transcribed and a loop represents a short, specific part of the DNA in a loop±chromomere complex. In situ nucleic acid hybridization is a means of locating specific gene sequences on chromosomes. Let us suppose that each loop represents `a gene.' The RNA that makes up the loop matrix, the attached nascent transcripts, will all be or include transcripts of
that `gene.' In effect, the loop is a large object, consisting of hundreds of RNA copies of the gene, all clustered at one position on the chromosome set. Isolate and purify the DNA of that gene, and label it in some way, and it will be easy to make it single-stranded and bind it specifically to the complementary singlestranded RNA attached to the lampbrush loop. The technique is known as DNA/RNA transcript in situ hybridization (DR/ISH). The end product of an experiment involving DR/ ISH is a preparation showing one or more pairs of loops with label distributed along their lengths. It is not uncommon in DR/ISH experiments to find loops that are labeled over only part of their lengths. This is evidence that the DNA sequence of a loop axis can and does change from place to place along the length of the loop. Wherever there are partially labeled loops, it is usual to find the same partially labeled loops, with precisely the same pattern of labeling, in every oocyte over quite a wide range of size and stage. So loops are permanent structures that transcribe from the same stretch of DNA axis throughout the entire lampbrush phase. DR/ISH experiments prove that highly repeated short DNA sequences, commonly referred to as `satellite' DNA, which could not possibly serve as a basis for transcription and translation into functional polypeptides, are abundantly transcribed on lampbrush loops along with more complex sequences that are definitely translated into functional proteins. The current hypothesis for LBC function is as follows. At the thin base of each loop or the start of each transcription unit there is a promoter site for a functional gene sequence. RNA polymerase attaches to this site and moves along the DNA, transcribing the sense strand of the gene and generating messenger RNA molecules that remain attached to the polymerase (Figure 5). In the lampbrush environment there are no stop signals for transcription, so the polymerases continue to transcribe past the end of the functional gene and into whatever DNA sequences lie `downstream' of the gene. This results in very long transcription units, very long transcripts, mixing of gene transcripts with nonsense transcripts in high molecular weight nuclear RNA, and lampbrush loops. This `read-through' hypothesis predicts that the number of functional genes that are expressed to form translatable RNAs may be expected to equal the number of transcription units that are active in a lampbrush set. The hypothesis says, in effect, that the only unusual feature of an LBC, and the very reason for the lampbrush form, is that once transcription starts it cannot stop until the polymerase meets another promoter that is already initiated or some condensed chromomeric chromatin that is physically impenetrable and untranscribable.
1076
L a m p b r u s h C h ro m o s o m e s
Figure 5 Transcription on a lampbrush chromosome loop where a gene (thick black line) is transcribed from its promoter (black flag) through to and past its normal stop signal (white flag) and into the normally nontranscribed DNA that lies downstream, thus generating very long transcription units with long transcripts that include RNA complementary to the sense strand of the gene (thick parts of the transcripts) and nonsense DNA that lies downstream of the gene (thin parts of the transcripts).
Evidence for the Read-Through Hypothesis for LBCs LBCs dissected directly into a solution of the enzyme deoxyribonuclease-1 (DNase-1) fall to pieces and their loops break into thousands of fragments. This does not happen with ribonuclease or proteases. If breakage of the chromosome axis and the loops by DNase is watched and timed and the number of breaks plotted against time on a log scale, the slope of the plot for the chromosome axis is 4 and that for the loops is 2. This supports the model in which the axis consists of two chromatids ± each a DNA double helix consisting of two nucleotide chains ± and the loop is part of one chromatid ± consisting of one double helix made up from two nucleotide chains. A later experiment used restriction enzymes that cleaved DNA only at places along the molecule where there was a particular short nucleotide sequence. If a loop consisted entirely of identical tandemly repeated DNA sequences, all with a particular restriction
enzyme recognition site, then the loop would be destroyed by that enzyme. If, on the other hand, the DNA sequences all lacked the enzyme recognition site, then the loop would be totally unaffected and would remain intact. An experiment was set up using five enzymes and the LBCs from N. viridescens. The control enzyme was deoxyribonuclease-1. DNase-1 and three of the restriction enzymes destroyed everything. One enzyme, HaeIII did likewise, except that it left one pair of loops completely intact. These HaeIII resistant loops were big ones, 100 mm long, equivalent to at least 300 000 nucleotides. Their unique resistance to HaeIII provided direct evidence that at least one pair of loops consisted of tandemly repeated short sequence DNA. At a later date, the effects of HaeIII were tested again, with appropriate controls, on the HaeIII resistant loops of N. viridescens. Breaks regularly occurred precisely at the thin beginnings of each loop, but the remainder of the loops remained intact, as would be predicted on the basis of the read-through hypothesis. The start of the transcription unit would be characterized by a long complex gene sequence that would almost inevitably include the HaeIII recognition site. The remainder of the loop would consist entirely of repeat sequences that lacked the HaeIII site.
Other Questions We Should Ask about Lampbrushes Only a small fraction of the entire DNA of a loop± chromomere complex forms the transcription unit that makes the loop. What about the rest of the DNA? Is the DNA segment that makes the loop preferentially selected for transcription the same piece at the corresponding locus in every egg of every individual of a particular species? This question may be approached experimentally. Why do loops have different morphologies that are heritable, locus-specific, and sometimes speciesspecific? The loop matrix is a site of processing, cleaving, and packaging of nuclear RNA, so most of the variation in gross structure may be expected to reflect different modes of binding and interaction involving quite a wide range of proteins and RNAs. Do LBCs look the same in all animals? They do not. The relative lengths of LBCs at the time of their maximum development are the same as the relative lengths of the corresponding mitotic metaphase chromosomes from the same species. The overall lengths of LBC are broadly related to genome size. Birds, with their notably small genomes, have extremely small, but nonetheless very beautiful, LBC that present many extraordinary and hitherto unexplained features.
L e a d er Pe p t i d e 1077 Some LBCs have long loops and others have very short ones. We have seen that the transcription units of LBCs are unusually long because they include interspersed repetitive elements of the genome. Structural genes in large genomes are more widely spaced than in small genomes as they are interspersed with noncoding DNA. One might therefore expect LBCs from large genomes to have longer loops (transcription units) than those of smaller genomes, and this is what has been observed. Many of the very long loops that we see in LBC from animals with large genomes show multiple, tandemly arranged thin±thick segments (transcription units). The individual transcription units within one loop can have the same or opposite polarities and can be of the same or different lengths (Figure 6). This observation suggests that it is really the transcription unit that is the ultimate genetic unit in an LBC and not the loop/chromomere complex, as was once thought. Why do LBCs exist at all? They are characteristic of eggs that develop quickly into complex multicellular organisms independently of the parent. A frog's egg is fertilized and develops into a complex tadpole within a few days. Much of the information and raw materials for this process are laid down during oogenesis through activity of LBCs and amplified ribosomal genes and the accumulation of yolk proteins imported from the liver. LBCs may therefore be regarded as an adaptive feature that has evolved to preprogramme the egg for rapid early development. The fact that they are not present in mammalian eggs could be regarded as an advanced feature that is consistent with the slow pace of mammalian development. A frog's egg, for example, will have completed gastrulation and the differentiation of its central nervous system and
embryonic axis by the time a human embryo has only reached the 8-cell stage. LBCs provide a uniquely powerful medium through which it has been possible to draw valid conclusions at the molecular level from observations and experiments carried out mainly with a light microscope. Their value extends into the fields of comparative molecular cytogenetics and systematics. Nowhere else is it possible to study genome structure, function, and diversity by actually looking at the genome itself with a light microscope. LBCs are technically challenging but not defeating. They are exceptionally beautiful to look at and fun to work with. Further information on these remarkable structures can be found in the literature listed in the Further Reading section below and on the internet.
Further Reading
Callan G (1986) Lampbrush Chromosomes. Berlin: SpringerVerlag. Macgregor HC (1993) An Introduction to Animal Cytogenetics. London: Chapman & Hall. Macgregor HC and Varley J (1988) Working with Animal Chromosomes. New York: John Wiley.
See also: Cytogenetics; Developmental Genetics
Late Genes E Kutter Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.0746
During viral infection, late genes are those that are transcribed after the commencement of viral DNA synthesis. The bulk of these encode either components of the capsid, proteins aiding in morphogenesis or DNA packaging, or proteins that are to be carried with the DNA in the capsid. See also: Virus
Leader Peptide Figure 6 The various arrangements of transcription units that actually occur on lampbrush chromosomes. The loop on the left comprises a single transcription unit. In the middle loop there are two transcription units of the same size and polarity. The right-hand loop has four transcription units of different sizes and different directions of polarity.
J Parker Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.0749
The term `leader peptide' (or, less commonly, `leader polypeptide') refers to a peptide encoded by a DNA