Proc. Nat. Acad. Sci. USA Vol. 72, No. 3, pp. 961-965, March 1975
Presynaptic Events in Meiocytes of Lilium longiflorum and Their Relation to Crossing-Over: A Preselection Hypothesis (heterochromatinization/nucleolar fusion/chromosome replication/chromosome alignment/recombination) H. STERN*, M. WESTERGAARDt, AND D. VON WETTSTEINtI * Department of Biology, University of California, San Diego, La Jolla, Calif. 92037; t Carlsberg Laboratory, Department of Physiology, DK-2500 Valby, Denmark; and I Institute of Genetics, University of Copenhagen, DK-1353, Copenhagen K, Denmark
Contributed by Mogens Westergaard, December 23, 1974 ABSTRACT We are proposing a "Preselection Hypothesis" to account for the regulation of crossing-over in eukaryotic organisms. The hypothesis characterizes meiosis in terms of three major physiological stages: (1) a presynaptic stage when pairs of homologous DNA stretches are selected so as to become trapped within the synaptinemal complex during synapsis, (2) an alignment of homologous chromosomes and stabilization of paired bivalents via the synaptinemal complex, and (3) a scission and rejoining of DNA stretches leading to the formation of chiasmata and crossovers. The hypothesis centers on the first stage and is based on evidence for the occurrence of significant cytological and biochemical changes prior to synapsis. The major feature of the hypothesis is that crossing-over occurs only in trapped DNA stretches. Thus, potential crossing-over sites, though not crossingover itself, are determined well before chromosomes pair. Since, to a large degree, crossovers are distributed randomly along the length of each chromosome, the preselection process must result in a random assortment of trapped DNA stretches, the assortment differing from one meiocyte to another.
The principal challenge in explaining crossing-over in eukaryotes is not so much the molecular mechanism of exchange between DNA strands as the mode of organization by which these exchanges are regularly effected in meiotic cells. Although prokaryotes provide a system of choice for probing the immediate events of DNA recombination, eukaryotes house facilitating mechanisms that are superimposed on the pure process of molecular recombination. The juxtapositioning of homologous chromosomes whose DNA lengths may exceed one meter, the apparent exclusion of sister-chromatid exchanges in the face of closely aligned sisters and homologs, the organization of homologous chromatids to accommodate four-strand crossing-over, the regulated distribution of exchanges as manifested in positive interference, and the restriction of the total process to differentiated meiocytes are specific items in the complex calendar of events governing
meiotic recombination in eukaryotes. Considerations
Although some uncertainty exists about the precise time at which strand exchanges occur in relation to the formation of the synaptinemal complex (SC), there can be little doubt that DNA strands transverse the complex at crossover sites. Unless crossing-over precedes synapsis, a possibility that we summarily set aside, its occurrence must be restricted to situations in which homologous regions are closely juxtaposed
within the SC. Such situations, however, would only allow for a very small proportion of DNA to engage in crossing-over. This follows from the morphology of the pachytene bivalent. Generally, "the SC joins homologous chromosomes over their whole length" (1). However, in the three species so far analyzed, namely Neurospora crassa (2), Drosophila melanogaster (3), and Zea mays (4), the length ratios of SC to chromosome DNA were about 0.3%, 0.2%, and 0.015%, respectively. Whether the length of the SC is determined by chromosome size or whether the reverse is the case, the significant point is that a pachytene chromosome must be appreciably contracted inasmuch as it would, if extended, exceed SC length by more than two orders of magnitude. The mutual accessibility of homologous DNA stretches is limited not only because of DNA compaction but also because of the 1200 A gap between the chromosomes which is occupied by the central region of the SC (1). Since the I)NA traversing the gap is below the limit of detection, the fraction of the genome within the SC of any single meiocyte must be exceedingly small. Despite this, the number of crossovers per bivalent is fairly constant and their distribution, outside of constitutive heterochromatic regions, is approximately random. To reconcile these features of regularity and randomness with the minute fraction of the genome which is available for crossing-over at pachytene, we propose that within each meiocyte there is a preselection of homologous DNA stretches which ultimately become incorporated into the SC. There is a considerable body of evidence that perturbations of meiocytes by certain agents, if applied prior to or during synapsis, affects the frequency of crossing-over (5). Much of the past controversy regarding the time of crossing-over appears to have been based on the view that the time at which an agent is effective coincides with the time at which crossing-over occurs. To be sure, the evidence for assigning the time of crossing-over to pachytene is extensive and strong, but the evidence for an influence of presynaptic events on crossing-over is considerable (6, 7). It may be justly argued that the development of an organizational framework for crossing-over is as directly related to crossing-over as is the molecular event of crossing-over itself. The presynaptic stages in Lilium longiflorum One of the more general facts to emerge from recent cytological studies of premeiosis is that there is a characteristic succession of stages prior to the onset of chromosome synapsis. Changes in distribution patterns of nonpermanent heterochromatin,
Abbreviation: SC, synaptinemal complex.
961
962
Genetics: Stern et al.
Proc. Nat. Acad. Sci. USA 72 .
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FIGS. 1-5. Lilium meiocyte nuclei at different presynaptic stages. FIG. 1. G-1 from 11.1 mm bud (anther = 5.8 mm); FIG. 2. S from 12.0 mm bud (anther = 6.0 mm); FIG. 3. G-2 from 12.1 mm bud (anther = 6.4 mm). The DNA contents were measured by Feulgen microspectrophotometry (H. Doll, B. Farestveit, and M. Westergaard, unpublished). FIG. 4. G-i; FIG. 5. Late leptotene. Figs. 1-3 are Feulgen squashes (X 1200); Figs. 4 and 5 are orcein squashes (X 2400).
in the shape and number of nucleoli, and in the morphology of the nuclear envelope appear to be widespread in occurrence and presumably have a functional significance (8-11). We have made a detailed cytological analysis of the presynaptic changes in a clone of Lilium longiflorum grown in the Stockholm phytotron. The appearance of the nucleus at each of several presynaptic stages is illustrated in Figs. 6-12. The procedure used in identifying individual stages is given in the legend, and is based on the correlation between bud or
anther length and meiotic stage (12). The presence of a slight developmental gradient between tip (the oldest meiocytes) and base (the youngest meiocytes) within an anther permits a more detailed resolution of the temporal sequence involving stages of short duration. We shall discuss the cytology in relation to the premeiotic stages, G-1, S, and G-2; and also to the presynaptic meiotic stages of early, mid, and late leptotene. Although the transitions between the subdivisions of leptotene are not sharp, it will be seen that they
Proc. Nat. Acad. Sci. USA 72
(1975)
Presynaptic Events in Meiosis
963
A, 9s t JoWO t
Presynaptic Events in Meiocytes of Lilium longiflorum and Their Relation to Crossing-Over: A Preselection Hypothesis (heterochromatinization/nucleolar fusion/chromosome replication/chromosome alignment/recombination) H. STERN*, M. WESTERGAARDt, AND D. VON WETTSTEINtI * Department of Biology, University of California, San Diego, La Jolla, Calif. 92037; t Carlsberg Laboratory, Department of Physiology, DK-2500 Valby, Denmark; and I Institute of Genetics, University of Copenhagen, DK-1353, Copenhagen K, Denmark
Contributed by Mogens Westergaard, December 23, 1974 ABSTRACT We are proposing a "Preselection Hypothesis" to account for the regulation of crossing-over in eukaryotic organisms. The hypothesis characterizes meiosis in terms of three major physiological stages: (1) a presynaptic stage when pairs of homologous DNA stretches are selected so as to become trapped within the synaptinemal complex during synapsis, (2) an alignment of homologous chromosomes and stabilization of paired bivalents via the synaptinemal complex, and (3) a scission and rejoining of DNA stretches leading to the formation of chiasmata and crossovers. The hypothesis centers on the first stage and is based on evidence for the occurrence of significant cytological and biochemical changes prior to synapsis. The major feature of the hypothesis is that crossing-over occurs only in trapped DNA stretches. Thus, potential crossing-over sites, though not crossingover itself, are determined well before chromosomes pair. Since, to a large degree, crossovers are distributed randomly along the length of each chromosome, the preselection process must result in a random assortment of trapped DNA stretches, the assortment differing from one meiocyte to another.
The principal challenge in explaining crossing-over in eukaryotes is not so much the molecular mechanism of exchange between DNA strands as the mode of organization by which these exchanges are regularly effected in meiotic cells. Although prokaryotes provide a system of choice for probing the immediate events of DNA recombination, eukaryotes house facilitating mechanisms that are superimposed on the pure process of molecular recombination. The juxtapositioning of homologous chromosomes whose DNA lengths may exceed one meter, the apparent exclusion of sister-chromatid exchanges in the face of closely aligned sisters and homologs, the organization of homologous chromatids to accommodate four-strand crossing-over, the regulated distribution of exchanges as manifested in positive interference, and the restriction of the total process to differentiated meiocytes are specific items in the complex calendar of events governing
meiotic recombination in eukaryotes. Considerations
Although some uncertainty exists about the precise time at which strand exchanges occur in relation to the formation of the synaptinemal complex (SC), there can be little doubt that DNA strands transverse the complex at crossover sites. Unless crossing-over precedes synapsis, a possibility that we summarily set aside, its occurrence must be restricted to situations in which homologous regions are closely juxtaposed
within the SC. Such situations, however, would only allow for a very small proportion of DNA to engage in crossing-over. This follows from the morphology of the pachytene bivalent. Generally, "the SC joins homologous chromosomes over their whole length" (1). However, in the three species so far analyzed, namely Neurospora crassa (2), Drosophila melanogaster (3), and Zea mays (4), the length ratios of SC to chromosome DNA were about 0.3%, 0.2%, and 0.015%, respectively. Whether the length of the SC is determined by chromosome size or whether the reverse is the case, the significant point is that a pachytene chromosome must be appreciably contracted inasmuch as it would, if extended, exceed SC length by more than two orders of magnitude. The mutual accessibility of homologous DNA stretches is limited not only because of DNA compaction but also because of the 1200 A gap between the chromosomes which is occupied by the central region of the SC (1). Since the I)NA traversing the gap is below the limit of detection, the fraction of the genome within the SC of any single meiocyte must be exceedingly small. Despite this, the number of crossovers per bivalent is fairly constant and their distribution, outside of constitutive heterochromatic regions, is approximately random. To reconcile these features of regularity and randomness with the minute fraction of the genome which is available for crossing-over at pachytene, we propose that within each meiocyte there is a preselection of homologous DNA stretches which ultimately become incorporated into the SC. There is a considerable body of evidence that perturbations of meiocytes by certain agents, if applied prior to or during synapsis, affects the frequency of crossing-over (5). Much of the past controversy regarding the time of crossing-over appears to have been based on the view that the time at which an agent is effective coincides with the time at which crossing-over occurs. To be sure, the evidence for assigning the time of crossing-over to pachytene is extensive and strong, but the evidence for an influence of presynaptic events on crossing-over is considerable (6, 7). It may be justly argued that the development of an organizational framework for crossing-over is as directly related to crossing-over as is the molecular event of crossing-over itself. The presynaptic stages in Lilium longiflorum One of the more general facts to emerge from recent cytological studies of premeiosis is that there is a characteristic succession of stages prior to the onset of chromosome synapsis. Changes in distribution patterns of nonpermanent heterochromatin,
Abbreviation: SC, synaptinemal complex.
961
962
Genetics: Stern et al.
Proc. Nat. Acad. Sci. USA 72 .
111111111111111111W.-W
*4
*
(197-6)
J6,
7 q 4
IF 1
4 ~ ~
f.
~
~
~
O
A.
:W
I _ A_
* 1
a
A.
2
3
alt.
FIGS. 1-5. Lilium meiocyte nuclei at different presynaptic stages. FIG. 1. G-1 from 11.1 mm bud (anther = 5.8 mm); FIG. 2. S from 12.0 mm bud (anther = 6.0 mm); FIG. 3. G-2 from 12.1 mm bud (anther = 6.4 mm). The DNA contents were measured by Feulgen microspectrophotometry (H. Doll, B. Farestveit, and M. Westergaard, unpublished). FIG. 4. G-i; FIG. 5. Late leptotene. Figs. 1-3 are Feulgen squashes (X 1200); Figs. 4 and 5 are orcein squashes (X 2400).
in the shape and number of nucleoli, and in the morphology of the nuclear envelope appear to be widespread in occurrence and presumably have a functional significance (8-11). We have made a detailed cytological analysis of the presynaptic changes in a clone of Lilium longiflorum grown in the Stockholm phytotron. The appearance of the nucleus at each of several presynaptic stages is illustrated in Figs. 6-12. The procedure used in identifying individual stages is given in the legend, and is based on the correlation between bud or
anther length and meiotic stage (12). The presence of a slight developmental gradient between tip (the oldest meiocytes) and base (the youngest meiocytes) within an anther permits a more detailed resolution of the temporal sequence involving stages of short duration. We shall discuss the cytology in relation to the premeiotic stages, G-1, S, and G-2; and also to the presynaptic meiotic stages of early, mid, and late leptotene. Although the transitions between the subdivisions of leptotene are not sharp, it will be seen that they
Proc. Nat. Acad. Sci. USA 72
(1975)
Presynaptic Events in Meiosis
963
A, 9s t JoWO t
