Heredity66 (1991) 453—458 Received 77 September 7990 Genetical Society of Great Britain

Positional control of chiasma distribution in the house mouse. Chiasma distribution in mice homozygous and heterozygous for an inversion in chromosome I

IVAN P. GORLOV, TATYANA YU. LADYGINA, OLEG L. SEROV & PAVEL M. BORODIN Institute of Cytology and , Academy of Sciences of the USSR, Siberian Department, Novosibirsk 630090, USSR

Anexamination of the chiasma distribution in chromosome 1 of male mice homozygous and heterozygous for a distal inversion In( 1) 1 2Rkandin normal males was carried out. No differences in chiasma distribution were found between homozygotes for the inversion and homozygotes for normal chromosome 1. A significant decrease in the frequency of bivalents bearing chiasmata in the pretelomeric region was found in heterozygotes. This, in its turn, produced a redistribution of chiasmata in the proximal non-inverted part of bivalent 1. These results could be interpreted as evidence for positional control of the chiasma distribution pattern: the distance of certain parts of the chromosome from the telomere and chiasmata interference are more important for determina- tion of the chiasma frequency in a given region than its genetic content.

Keywords:chiasma,chromosome 1 inversion, house mouse.

Introduction et a!., 1987). The latter observation could be consid- ered as a reflection of the second limitation of chiasma Thedistribution of chiasmata within bivalents is, with distribution within a bivalent, namely, chiasma interfer- rare exceptions (Strickland, 1958; Olson et a!., 1978), ence. This can be inferred from comparisons of chi- never random (Jones, 1986). If it were random, asma distributions in bivalents with one or two chiasmata would occur with equal probability in all chiasmata. These distributions differ considerably, so parts of the bivalent and the position of two chiasmata that single chiasmata tend to occupy rather central in the same bivalent would be independent of each positions in bivalents while double chiasmata occupy other. Jones (1987) mentions two principal identifiable relatively more proximal and distal locations. limitations of chiasma distribution within bivalents: Studies of the molecular mechanisms of crossing- localization and interference. over have revealed some hot spots of recombination There is an obvious tendency for chiasmata to arise determined by the primary nucleotide sequence in preferentially or exclusively in certain regions of the DNA (Steinmetz et al., 1987). A case was found where bivalent (Darlington, 1931; Mather, 1937, 1938; a single base substitution in yeast led to a 10-fold Henderson, 1963; Fox, 1973). Extreme subcentro- increase in recombination frequency (Ronticelli et a!., metric and/or subtelomeric localization of chiasmata 1988). It has also been suggested that the localization has been found in some species (Callan & Perry, 1977). of minisatellite DNA, which is homologous to the Chi- However, many species demonstrate not a qualitative, sites of prokaryotes, plays an important role in the but a quantitative localization of chiasmata in the sense control of chiasma distribution within bivalents (Jar- that they are more frequent in some regions than in man & Wells, 1989). In situ hybridization on human others. For example, long bivalents (1—3) of the house meiotic metaphase I chromosomes, using the labelled mouse display a tendency to subcentromeric and minisatellite core sequence, showed clusterings of subtelomericlocalization,while the remaining autoradiographic grains principally at or around chias- bivalents show predominantly subtelomeric localiza- mata (Chandley & Mitchell, 1988). tion. Relatively few chiasmata arise in the interstitial The aim of this study was to investigate the factor(s) part of the bivalents (Maudlin & Evans, 1980; Gorlov controlling chiasma localization in mice. More impor- 453 454 I. P. GORLOV ETAL. tantly does the genetic content of a definite region (i.e. nique to identify subcentromeric C-heterochromatin in localization of certain DNA sequence) or the position chromosome 1 (Sumner, 1972). of this region with respect to centromere and telomere Well-spread diplotene and diakinesis nuclei (Fig. 1) determine its chiasma frequency? were traced using a drawing apparatus. Each bivalent of chromosome 1, marked by a small precentromeric C-band, was subdivided into 10 equal parts. The Materialsand methods frequency of bivalents with chiasmata in each part was Maleand female mice homozygous for a paracentric calculated. Differences between individual males inversion in chromosome 1 (In(1) l2Rk—1n12) were within genotypes in chiasma distribution were tested gifts of Dr T. H. Roderick from the Jackson Labora- according to the methods used by Laurie & Jones tory, Bar Harbor, Maine, USA. (1981). The distribution patterns of chiasmata between High-resolution G-band staining (Ikeushi, 1984) was genotypes were compared statistically by Chi-square used for the precise localization of the breakpoint of method for two empirical distributions with 9 d.f. this inversion on the cytological map of mitotic (Kendall & Stuart, 1973). Differences in chiasma chromosome 1. Surface-spread preparations of frequency (f)betweencertain regions of the bivalent synaptonemal complexes (SC) from the spermatocytes were tested using F-criterium after p-transformation, of males heterozygous for In 12 were made according with p =2arcsin fj(Fisher& Yates, 1938; Auscombe, to the method advanced by Solari (1980), stained with 1948). AgNO3 (Howell & Black, 1980) examined and photo- graphed with an electron microscope JEM (Jeol, Results Japan) at 80 kV. The locations of the endpoints of inside and outside the inversion loops were presented as proportions of the distance between the Cytological mapping of the in version centromere and the corresponding point to the mean So far inversion 1n12 has been localized only on the length of the two lateral elements of the given SC. genetic map (Roderick, 1981). The proximal break- These data were used to localize the breakpoints of the point was found to be situated in the middle of the map inversion on the map of the SC. (45 cM from the centromere), and the distal breakpoint The F1 heterozygotes ml 2Rk/ + were produced by is located near the telomere. According to the pattern crossing Inl2Rk homozygotes with a CBkLac strain. of high-resolution banding (Fig. 2), we localized the The latter possessed chromosome 1 marked by a small proximal breakpoint between subbands El and E3 block of subcentromeric C-heterochromatin (Forejt, and the distal breakpoint at band H6. The distances of 1973), which was used to identify the bivalent 1 in diplotene—diakinesis spreads. Homozygotes and heterozygotes for 1n12, as well as normal homo- zygotes, were obtained from F2 crosses among F1 prog- eny. Karyotypes of the individuals were identified by a I 4 biochemical marker, peptidase 3. The gene for pepti- S dase 3 (Pep-3) has been localized inside the inverted segment of chromosome 1 (Roderick et at., 1981). S 1n12 contained allele Pep-3' (slow variant). Normal chromosome 1 derived from CBA/Lac strain carried • allele Pep3b (fast variant). Electrophoretic separation and histochemical staining were performed as * s*It described previously (Rubtsov etat., 1982). F2 homozygotes for the inversions, selected for the analy- sis of chiasma distribution, also possessed a small * S C-positive block. They were crossovers between the centromere and the inversion. p Three, 3-month-old males of each genotype were used to study chiasma distribution. Diplotene—diakine- 4* sis chromosome spreads were prepared after hypo- tonic treatment and methanol:acetic acid (3:1) fixation by a routine air drying technique (Evans et at., 1964). Fig. 1 Diakinesis in a male mouse heterozygous for the Slides were processed by the C-band staining tech- small C-positive block in chromosome 1 (arrowed). Fig. 3Differentsynapticconfigurations ofthechromosome from thecentromeretoproximalpointofasynapsis figuration foundin8.052.21percentofthenucleiis ments oftheaxesandwithasynapticzonein incomplete inversionloopwithunpaireddistalseg- frequent configuration(34.893.89percent)wasan tion (2.011.10percent)ofthenuclei.Amore paired endsofaxes(Fig.3a)werefoundinasmallfrac- paired intheinvertedsegment.Theremainingnuclei lents. Bivalent1inthesenucleiwasheterologously one-half ofthenuleishowedallstraight-pairedbiva- somes fromthreeheterozygousmales.Approximately nuclei withfullparingofallnon-invertedchromo- total of149silver-stainedsurface-spreadpachytene synapsis, T= endpoint ofstraightsynapsis,D = micrograph. Silverstaining.C= 1 inmalemiceheterozygousfor In(1)12Rk.Electron study ofSCwastocomparethesynapticpattern where (Borodineta!., for inversionsinchromosome1arepublishedelse- synaptic adjustmentinsingleanddoubleheterozygotes diakinesis bivalent. segments 6and10respectivelyofthediplotene— mitotic mapofchromosome1.Theycorrespondto positions ofthebreakpointsinversionon figuration awas0.950.02.Thisagreeswiththe centromere tothedistalpointofasynapsisincon- in configurationsaandbwas0.520.02,fromthe meric endswereunpaired.TherelativelengthofSC shown atFig.3c.Ithadnoinversionloopbutitstelo- proximal partoftheloop(Fig.3b).Thethirdcon- figuration. Incompleteinversionloopswithfully showed threetypesofheteromorphicsynapticcon- respectively. these pointsfromthecentromerewere0.57and0.98, show breakpoints.(a)Normalchromosome,(b)In(1)12Rk. mitotic metaphasechromosome1.0-bandstaining.Arrows Fig. 2Microphotographandschematicpresentationof Synaptonemal complexes(SCs)werestudiedina More detailsonthechromosomepairingand telomere.

I I 1990). .C)flrTI () > centromere, The mainpurposeofthis distal a endpoint ofstraight P= proximal

II II mIm I 1101 C) w CHIASMA DISTRIBUTIONINTHEHOUSEMOUSE455 y_ 'i.k- 2 C T. TJA? 456 I. P. GORLOV ETAL.

chromosome 1 with the pattern of chiasma distribution heterozygous and homozygous for 1n12 males were along bivalent 1 in heterozygotes for the inversion. 249, 210 and 305, respectively. There were no differ- ences between individual males within genotypes in chiasma distribution (chi squares for normal, hetero- Chiasmadistribution zygous and homozygous were 26.7, 24.7, 20.0, Thenumbers of C-band diplotene and diakinesis respectively, P> 0.05), thus the data within the geno- nuclei scored for chiasma distribution from normal, types were pooled. Figure 4 shows the distributions of chiasmata along bivalent 1 in the three genotypes. (a) The chiasma distribution in normal and homo- zygous for 1n12 males (Fig. 4a,b) was identical (chi -I- square=3.6, P<0.5), i.e. inversion of the chromo- C) somal material did not produce any inversion of the aa' 40 C pattern of chiasma distribution. Homozygotes did not C) 1 C.) differ from normal males in the total chiasma number a. (1.62 0.03 and 1.67 0.03, respectively, t=1.18, 20 P>0.05). As the breakpoints of the inversion were localized inside segments 6 and 10, it would be incorrect to com- pare the chiasma frequency within these regions flH11LL between the normal and inversion homozygotes. Chiasma frequency in region 7 in normal mice was significantly lower than in region 9 (F=4.2, P<0.05). (b) On being transferred to the pretelomeric region 9 in the inversion homozygote, however, it displayed as high a frequency as region 9 in the normal homo- a'C) C) zygotes and vice versa (F= 0.96, F> 0.01). C C) Chiasma distribution in heterozygotes (Fig. 4c) C.) C) differed drastically from those in normal and inversion a. homozygotes (Chi square 94.7, P < 0.01). Chiasmata 20 occurred in the telomeric region in heterozygotes three times less frequently than in the same region in homo- zygotes. This is not surprising because, as we have shown, this region was unpaired in about 40 per cent of pachytene nuclei. Weak and unstable synapsis inside the inversion loop and a high incidence of hetero- (c) synapsis led to a decrease in chiasma frequency in the inverted region. As a result, the total chiasma number C) per bivalent was much smaller in heterozygotes (1.21 0.03) than in homozygotes and normal males (t=10.37,P<0.01). A crossover suppression in the distal part of the bivalent produced chiasma redistribu- tion in the proximal non-inverted part. The subcentro- I meric peak characteristic of homozygotes was smoothed out in heterozygous males.

Discussion 1H3 45 67 8 910 Theaim of this study was to investigate which of the Fig. 4Chiasmafrequency distribution in the ten segments of factors that determine the probability of chiasma the bivalent 1 (percentages of bivalents with chiasma in the occurrence in a definite region of the chromosome are segment). (a) Homozygotes for the normal chromosome 1, the more important, the genetic content of the region (b) homozygotes for In( 1)1 2Rk, (c) heterozygotes for In( 1) (i.e. localization of certain DNA sequence) or its posi- l2Rk. tion with respect to the centromere and telomere? CHIASMA DISTRIBUTION IN THE HOUSE MOUSE 457

Inversion homozygotes represent a convenient follows a linear and temporal sequence from the fixed model for the examination of this problem. Different point in a bivalent. He takes the centromere as the parts of the region that are confined by the breakpoints starting point. A number of data on chiasma distribu- significantly differ from each other in chiasma tion in different species have been interpreted in terms frequency. Chiasmata rarely occur near the proximal of this model. The only differences in this interpreta- breakpoint (region 7) and occur more often in the tion concern the consideration of telomeres rather than subtelomeric part (region 9) of the normal bivalent. If centromeres as the starting point (see Jones, 1987, for the chiasma localization in any part of any chromo- a review). some is determined predominantly by the genetic It has been suggested that chiasma distribution can content of this part, the pattern of chiasma distribution be inferred from pairing characteristics such as the in the bivalent 1 in homozygotes for the inversion is position of the initiation point and the rate of synaptic inverted. We have not found, however, any differences progression (Moens, 1969; Sybenga, 1975). A close in chiasma distribution patterns between normal and correlation between pairing frequencies of definite seg- inversion homozygotes. On the other hand, we have ments and the recombination frequencies in respective found an altered pattern of the chiasma distribution in regions has been demonstrated (Maguire, 1966, 1977; the proximal non-inverted part of bivalents in hetero- Parker etal., 1982; Parker, 1987). zygotes, while the order of genes in this region in In our experiment a correlation between the pairing homozygotes and in heterozygotes was the same. failure in the telomeric region of chromosome 1 in Similar results were found in a study of chiasma inversion heterozygotes and a substantial decrease in distribution along bivalent 1 of two chromosomally the frequency of chiasmata in the pretelomeric segment differentiated taxa of the grasshopper Caledia captiva was also shown. The leading role of the mammalian (Shaw & Wilkinson, 1980). These taxa were supposed telomere as the starting point of pairing has been to differ by a pericentric inversion in chromosome 1, demonstrated in different studies (Speed, 1989). This transforming anacrocentric chromosome of may be the reason why the segment of the chromosome 'Torresian' variety into a metacentric chromosome of located near the starting point has the highest chiasma 'Moreton' variety. Nevertheless, chiasma distributions frequency (Maudlin & Evans, 1980; Gorlov et a!., in both homozygotes were almost identical to a 1987). Recombination frequency in the distal part of proximal/distal pattern of chiasma localization. The the bivalent decreases in proportion to the distance distribution pattern of chiasma in the non-inverted part between the telomere and the segment. We have shown of chromosome 1 in heterozygotes was very different that this is true regardless of its genetic content. from that observed in either parent. From this point of view it is interesting to reconsider These results can be interpreted as evidence of the the evolutionary role of inversions. In addition to the positional control of the chiasma distribution pattern. classical interpretation of inversions as tools of con- The distance from any part of the chromosome to the servation of co-adaptive gene complexes (Dobzhansky, telomere and the action of chiasma interference are 1970) and as factors that affect gene expression more important for the determination of chiasma through the position effect, we would like to emphasize frequency in the given region than the genetic content their 'recombinational position effect'. Homozygotes of this region. for an inversion may differ from normal homozygotes This is not to deny the existence of a local site- with respect to recombination between certain genes of specific control mechanism. Thousands of potential the chromosome. It can affect the rate of the combina- sites of recombination exist as evidenced by the data tive variation for certain traits and, as a result, deter- obtained in molecular studies (Stern & Hotta, 1987). mine the probability of fixation of 'normal' or inverted They are dispersed evenly throughout the genome and chromosome. vary in their recombinogenic activity. They are respon- sible for the fine control of recombination, however, Acknowledgements the positional control of chiasma distribution eclipses Weare extremely grateful to Dr L. V. Vysotskaya and these microvariations. Chiasma determination appears Dr B. F. Chadov for their helpful comments on the to be a characteristic not of a particular site but of the manuscript, and to Mrs Marina I. Rodionova for position of the segment on the chromosome. The technical assistance. subtelomeric segments of any site composition are characterized by higher chiasma frequencies than any References other segments of the chromosome. In general, our data fit Mather's (1937, 1938) AuSCOMBE,r. j.1948.The transformation of Poisson, binomial sequential model, in which chiasma determination and negative-binomial data, Biometrics, 35, 246—254. 458 I. P. GORLOV ETAL.

BORODIN, P. M., GORLOV, I. P. AND LADYGINA, T. 't'u. 1990. Synapsis MATHER, K. 1937. The determination of position in crossing- in single and double heterozygotes for partially over- over. II. The chromosome length-chiasma frequency rela- lapping inversions of chromosome 1 of the house mouse. tion. Cytologia Fujii Jubilee, pp. 5 14—526. Chromosoma, 99, 365—370. MATHER, K. 1938. Crossing-over. Biol. Rev., 13, 252—292. CALLAN, 1-1. 0. AND PERRY, P. E. 1977. Recombination in male MAUDLIN, I. AND EVANS, E. p, 1980. Chiasma distribution in and female meiocytes contrasted. Philos. Trans. R. Soc. mouse oocytes during diakinesis. Chromosoma, 80, Lond.,B277,227—233. 259—262. CHANDLEY, A. C. AND MITCHELL, A. M. 1988. Hypervariable mini- MOENS, p 1969. The fine structure of meiotic chromosome satellite regions are sites for crossing-over at in polarization and pairing in Locusta migratoria Spermato- man. Cytogenet. Cell Genet., 48,152—155. cytes. Chromosoma, 28, 1—25. DARLINGTON, C. D. 1931. Meiosis. Biol. Rev. Camb. Philos. OLSON, L. W., EDEN, U., EGEL-MITANI, M. AND EGEL, R. 1978. Soc., 6,221—264. Asynaptic meiosis in fission yeast? Hereditas, 89, DOBZHANSKY, T. 1970. Genetics of the Evolutionary Process. 189—199. Columbia University Press, New York. PARKER, J. s. 1987. Increased chiasma frequency as a result of EVANS, E. P., BRECKON, 0. AND FORD, C. D. 1964. An air-drying chromosome rearrangement. Heredity, 58, 87—94. method for meiotic preparations from mammalian testes. PARKER, J. S., PALMER, R. W., WHITEHORN, M. A. F. AND EDGAR, L. A. Cytogenetics, 3, 289—299. 1982. Chiasma frequency effects of structural chromo- FISHER, R. A. AND YATES, F. 1938. Statistical Tables for Biologi- some change. Chromosoma, 85, 673—686. cal, Agricultural and Medical Research. Oliver and Boyd RODERIcK,T. H. 1981. Map of inversions. In: Green, M. C. (ed.) Ltd, Edinburgh. Genetic Variants and Strains of the Laboratory Mouse. FOREJT, j.1973.Centromeric heterochromatin polymorphism Gustav Fisher Verlag, Stuttgart, pp. 366—368. in the house mouse. Evidence from inbred strains and RODERICK, T. H., STAATS, J. AND WOMACK, J. E. 1981. Strain distri- natural populations. Chromosoma, 43, 187—20 1. bution of polymorphic variants. In: Green, M. C. (ed.) FOX, D. p 1973. The control of chiasma distribution in the Genetic Variants and Strains of the Laboratory Mouse. locust, Schistocerca gregaria (Forskal). Chromosoma, 65, Gustav Fisher Verlag, Stuttgart, pp. 377—397. 247-267. RONTICELLI, A. 5., SENA, E. P. AND SMITH, G. R. 1988. Genetic and GORLOV, I. P., AGULNIK, A. I. AND AGULNIK, s. 1. 1987. Localization physical analysis of the M26 recombination hot spot of of house mouse genes on chromosomes via comparison of Schizosaccharomyces pombe. Genetics, 119,491—497. the genetic map and the chiasmata distribution. Genetika, RUBTSOV, N. B., GRADOV, A. A. AND SEROV, 0. L. 1982. Chromo- 23, 63—70 (in Russian). some localization of the loci GOT1, PP, NP, SOD 1, PEPA HENDERSON, S. A. 1963. Chiasma distribution at diplotene in a and PEPC in the American mink (Mustela vison). Teor. locust. Heredity, 18, 173—190. AppI. Genet., 63, 331—336. HOWELL, W. M. AND BLACK, D. A. 1980. Controlled silver-staining SHAW, D. D. AND WILKINSON, p, 1980. Chromosome differentia- of nucleolus organizer-regions with protective colloidal tion, hybrid breakdown and maintenance of a narrow developer: a 1-step method. Experientia, 36, 1014—1015. hybrid zone in Caledia. Chromosoma, 80, 1—32. IKEuCHI, i'. 1984. Inhibitory effect of ethidium bromide on SOLARI, A. J. 1980. Synaptonemal complexes and associated mitotic chromosome condensation and its application to structures in microspread human spermatocytes. Chromo- high-resolution chromosome banding. Cytogenet. Cell soma, 81, 315—337. Genet., 38,56—61. SPEED, R. El. 1989. Heterologous pairing and fertility in JARMAN, A. R. AND WELLS, R. A. 1989. Hypervariable mini- humans. In: Gillies, C. B. (ed.) Fertility and Chromosome satellites: recombinators or innocent bystanders? Trends Pairing: Recent Studies in Plants and Animals. CRC Press, Genet., 5,367—371. Florida, pp. 1—35. JONES, 0. H. 1986. The control of chiasma distribution. Symp. STEINMETZ, M., UEMAT5U, Y. AND FISHER, L. K. 1987. Hotspots of Soc. Exp. Biol., 38, 29 1—320. homologous recombination in mammalian genomes. JONES, 0. H. 1987. Chiasmata. In: Moens, P. B. (ed.) Meiosis. Trends Genet., 3, 7—10. Academic Press, London, pp. 2 13—244. STERN, H. AND H0TrA, y, 1987. The biochemistry of meiosis. In: KENDALL, M. 0. AND STUART, E. 1973. The Advanced Theory of Moens, P. B. (ed.) Meiosis. Academic Press, London, pp. Statistics. Griffin, London. 303—33 1. LAURIE, D. A. AND JONES, G. H. 1981. Inter-individual variation STRICKLAND, W. N. 1958. An analysis of interference in Asper- in chiasma distribution in Chorthippus brunneus (Ortop- gil/us nidulans. Proc. R. Soc., B149, 82—111. tera: Acaridae). Heredity, 47, 409—4 16. SUMNER, A. T. 1972. A simple technique for demonstrating MAGUIRE, M, P 1966. The relationship of crossing over to centromeric heterochromatin. Expl Cell Res., 75, chromosome synapsis in a short paracentric inversion. 304—306. Genetics, 53, 1071—1077. SYBENGA, J. 1975. Meiotic configurations. In: Frankel, R. (ed.) MAGUIRE, M. P. 1977. pairing. Phil. Monographs on Theoretical and Applied Genetics 1, Trans. R. Soc. Lond., B277, 245—258. Springer-Verlag, Berlin, pp. 1—125.