Super-stretched pachytene chromosomes for fluorescence in situ hybridization mapping and immunodetection of DNA methylation

Meiotic pachytene chromosome-based fluorescence in situ hybridization (FISH) mapping is one of the most important tools in plant molecular cytogenetic research. Here we report a simple technique that allows stretching of pachytene chromosomes of maize to up to at least 20 times their original size. A modified Carnoy's II fixative (6:1:3 ethanol:chloroform:acetic acid) was used in the procedure, and proved to be key for super-stretching of pachytene chromosomes. We demonstrate that super-stretched pachytene chromosomes provide unprecedented resolution for chromosome-based FISH mapping. DNA probes separated by as little as 50 kb can be resolved on super-stretched chromosomes. A combination of FISH with immunofluorescent detection of 5-methyl cytosine on super-stretched pachytene chromosomes provides a powerful tool to reveal DNA methylation of specific chromosomal domains, especially those associated with highly repetitive DNA sequences.     

A tandemly repeated DNA sequence is associated with both knob-like heterochromatin and a highly decondensed structure in the meiotic pachytene chromosomes of rice

Highly repetitive tandem DNA sequence repeats are often associated with centromeric and telomeric regions of eukaryotic chromosomes. The rice tandem repeat Os48 is organized as long arrays of a 355 bp monomer and is mainly located in the telomeric regions. The chromosomal locations of the Os48 sequence were determined by fluorescence in situ hybridization (FISH) on rice pachytene chromosomes. The majority of the Os48 loci are associated with brightly 4',6-diamidino-2-phenylindole (DAPI)-stained and knob-like heterochromatin in rice pachytene chromosomes. As with other DNA sequences located in the heterochromatic regions, the cytosines of the CG and C(A/T)G sites within the Os48 repeat are heavily methylated. Surprisingly, a proportion of the FISH signals are highly decondensed and deviate significantly from the DAPI-stained periphery of the pachytene chromosomes. This highly decondensed chromatin structure has not been reported in pachytene chromosomes prepared from alcohol/acid-fixed meiotic samples in any other eukaryotic species. The condensation of the Os48 sequences is dynamic during prophase I of meiosis. The FISH signals derived from the Os48 repeat progress from a condensed configuration between leptonema and early pachynema into a decondensed structure from middle pachynema to diakinesis, and then return to a condensed form at metaphase I.     

Morphology of a human-derived YAC in yeast meiosis

In meiosis of human males DNA is packaged along pachytene chromosomes about 20 time more compactly than in meiosis of yeast. Nevertheless, a human-derived yeast artificial chromosome (YAC) shows the same degree of compaction of DNA as endogenous chromosomes in meiotic prophase nuclei of yeast. This suggests that in yeast meiosis, human and yeast DNA adopt a similar organization of chromatin along the pachytene chromosome cores. Therefore meiotic chromatin organization does not seem to be an inherent chromosomal property but is governed by the host-specific cellular environment. We suggest that there is a correlation between the less dense DNA packaging and the increased rate of recombination that has been reported for human-derived YACs as compared with human DNA in its natural environment.     

番茄的CPD带型和45S rDNA位点的鉴别

采用CPD（PI和DAPI组合）染色对番茄减数分裂粗线期和有丝分裂中期染色体进行了显带分析,随后用两种不同的45S rDNA克隆在相同的分裂相进行了荧光原位杂交定位分析。CPD染色在8条粗线期染色体上显示出了10条红色的CPD带纹,在6对有丝分裂中期染色体上显示出了12条CPD带纹。有丝分裂中期染色体上的CPD带纹与粗线期染色体上显著的带纹具有对应性。用改良的CPD染色程序清晰而稳定地显示出这些特征性的CPD带纹为番茄的染色体,特别是有丝分裂中期染色体提供了新的识别标记。用番茄的一个45S rDNA克隆进行的荧光原位杂交,不仅在位于2号染色体短臂的随体上显示了强的杂交信号,而且在粗线期染色体的5个CPD带区或有丝分裂中期染色体的4对CPD带区显示了弱的杂交信号。然而,用来自小麦的45S rDNA克隆pTa71进行的原位杂交却只在随体上显示了杂交信号。鉴于所用的两个45S rDNA克隆在序列上的差异,推断在番茄基因组中只有随体含有45S rDNA单位的编码区,即番茄只有一对45S rDNA位点。     

Direct FISH of 5S rDNA on tomato pachytene chromosomes places the gene at the heterochromatic knob immediately adjacent to the centromere of chromosome 1.

We describe a molecular cytogenetic procedure for high resolution physical mapping of DNA markers, an essential step toward construction of an integrated molecular-classical-cytological map. Tomato was selected as material because its pachytene chromosomes are amenable for study and because detailed molecular, classical, and cytological maps are available. Karyotyping of acetocarmine-stained pachytene chromosomes showing detailed cytogenetic landmarks was combined with direct FISH of the 5S rDNA gene. This enabled us to pinpoint the 5S rDNA gene to the first heterochromatic knob immediately adjacent to the centromere in the short arm of chromosome 1. Thus the position of the 5S rDNA gene on the molecular map was related to the position of the 5S rDNA on the cytogenetic map. The results also provide conclusive evidence of the location of a functional gene in the pericentric heterochromatic region, a rare event to date in plants. We conclude that karyotyping of pachytene chromosomes can be combined with FISH to map a DNA sequence to a cytogenetically defined region and to determine the chromatin origin of an expressed gene. Key words : direct fluorescence in situ hybridization, 5S rDNA, pachytene chromosomes, heterochromatic gene.     

In situ DNA sequence mapping with surface-spread mouse pachytene chromosomes

Surface-spread pachytene chromosomes are several times the length of metaphase chromosomes and the decondensed chromatin loops are attached to a well-defined axis (Weith and Traut, 1980). This arrangement permits detailed DNA sequence localization by in situ hybridization. We show that two probes to low-frequency repeated sequences (20 to 50 copies) which locate the centromere proximal in the mouse X metaphase chromosome between bands A1 and A3 (Disteche et al., 1985) and which map 5.5 cM apart (Disteche et al., 1989), hybridize to two distinct chromatin regions 3 to 5 microns apart on a 25 microns long pachytene X chromosome core.     

Differential elongation of autosomal pachytene bivalents related to their DNA content in human spermatocytes

The establishment of the complete karyotype of human pachytene spermatocytes reveals differences in stretching of chromosomes between meiosis and mitosis. Bivalents or specific regions of bivalents which exhibit many R-bands are particularly elongated. In mitotic chromosomes, the DNA contained in such bands is known to be early replicating. The study of variations in the total length and the centromeric index of bivalent 1 suggests that differential elongation of pachytene bivalents is a premeiotic event, taking place during the last DNA replication.     

A high-resolution karyotype of cucumber (Cucumis sativus L. 'Winter Long') revealed by C-banding, pachytene analysis, and RAPD-aided fluorescence in situ hybridization.

Using molecular cytogenetic DNA markers, C-banding, pachytene analysis, and fluorescence in situ hybridization (FISH), a high-resolution karyotype was established in the cucumber. C-banding showed distinct hetero chromatic bands on the pericentromeric, telomeric, and intercalary regions of the chromosomes. The C-banding patterns were also consistent with the morphology of 4'-6-diamino-2-phenylindole dihydrochloride (DAPI)-stained pachytene chromosomes. Two repetitive DNA fragments, CsRP1 and CsRP2, were obtained by PCR and localized on the mitotic metaphase and meiotic pachytene chromosomes. CsRP1 was detected on the pericentromeric heterochromatic regions of all chromosomes, except chromosome 1. CsRP2 was detected on 5 (chromosomes 1, 2, 3, 4, and 7) of 7 chromosomes. All homologous chromosome pairs could be distinguished by FISH using 2 RAPD markers. This is the first report on molecular karyotyping of mitotic and meiotic spreads of cucumber.     

Cytogenetics for the model system Arabidopsis thaliana

A detailed karyotype of Arabidopsis thaliana is presented using meiotic pachytene cells in combination with fluorescence in situ hybridization. The lengths of the five pachytene bivalents varied between 50 and 80 μm, which is 20–25 times longer than mitotic metaphase chromosomes. The analysis confirms that the two longest chromosomes (1 and 5) are metacentric and the two shortest chromosomes (2 and 4) are acrocentric and carry NORs subterminally in their short arms, while chromosome 3 is submetacentric and medium sized. Detailed mapping of the centromere position further revealed that the length variation between the pachytene bivalents comes from the short arms. Individual chromosomes were unambiguously identified by their combinations of relative lengths, arm-ratios, presence of NOR knobs and FISH signals with a 5S rDNA probe and chromosome specific DNA probes. Polymorphisms were found among six ecotypes with respect to the number and map positions of 5S rDNA loci. All ecotypes contain 5S rDNA in the short arms of chromosomes 4 and 5. Three different patterns were observed regarding the presence and position of a 5S rDNA locus on chromosome 3. Repetitive DNA clones enabled us to subdivide the pericentromeric heterochromatin into a central domain, characterized by pAL1 and 106B repeats, which accommodate the functional centromere and two flanking domains, characterized by the 17 A20 repeat sequences. The upper flanking domains of chromosomes 4 and 5, and in some ecotypes also chromosome 3, contain a 5S rDNA locus. The detection of unique cosmids and YAC sequences demonstrates that detailed physical mapping of Arabidopsis chromosomes by cytogenetic techniques is feasible. Together with the presented karyotype this makes Arabidopsis a model system for detailed cytogenetic mapping.     

A high-resolution karyotype of <Emphasis Type="Italic">Brassica rapa</Emphasis> ssp. <Emphasis Type="Italic">pekinensis</Emphasis> revealed by pachytene analysis and multicolor fluorescence in situ hybridization

A molecular cytogenetic map of Chinese cabbage (Brassica rapa ssp. pekinensis, 2n=20) was constructed based on the 4-6-diamino-2-phenylindole dihydrochloride-stained mitotic metaphase and pachytene chromosomes and multicolor fluorescence in situ hybridization (McFISH), using three repetitive DNA sequences, 5S rDNA, 45S rDNA, and C11-350H. The lengths of mitotic metaphase chromosomes ranged from 1.46 m to 3.30 m. Five 45S and three 5S rDNA loci identified were assigned to different chromosomes. The C11-350H loci were located on all the mitotic metaphase chromosomes, except chromosomes 2 and 4. The pachytene karyotype consisted of two metacentric (chromosomes 1 and 6), five submetacentric (chromosomes 3, 4, 5, 9 and 10), two subtelocentric (chromosomes 7 and 8), and one acrocentric (chromosome 2) chromosome(s). The mean lengths of ten pachytene chromosomes ranged from 23.7 m to 51.3 m, with a total of 385.3 m, which is 17.5-fold longer than that of the mitotic metaphase chromosomes. In the proposed pachytene karyotype, all the chromosomes of B. rapa ssp. pekinensis can be identified on the basis of chromosome length, centromere position, heterochromatin pattern, and the location of the three repetitive sequences. Moreover, the precise locations of the earlier reported loci of 5S rDNA, 45S rDNA, and Chinese cabbage tandem DNA repeat C11-350H were established using McFISH analysis. We also identified a 5S rDNA locus on the long arm of pachytene bivalent 7, which could not be detected in the mitotic metaphase chromosomes in the present and earlier studies. The deduced karyotype will be useful for structural and functional genomic studies in B. rapa.     

The pachytene chromosomes of maize as revealed by fluorescence in situ hybridization with repetitive DNA sequences

A repetitive DNA sequence, ZmCR2.6c, was isolated from maize based on centromeric sequence CCS1 of the wild grass Brachypodium sylvaticum. ZmCR2.6c is 309 bp in length and shares 65% homology to bases 421–721 of the sorghum centromeric sequence pSau3A9. Fluorescence in situ hybridization (FISH) localized ZmCR2.6c to the primary constrictions of pachytene bivalents and to the stretched regions of MI/AI chromosomes, indicating that ZmCR2.6c is an important part of the centromere. Based on measurements of chromosome lengths and the positions of FISH signals of several cells, a pachytene karyotype was constructed for maize inbred line KYS. The karyotype agrees well with those derived from traditional analyses. Four classes of tandemly repeated sequences were mapped to the karyotype by FISH. Repeats 180 bp long are present in cytologically detectable knobs on 5L, 6S, 6L, 7L, and 9S, as well as at the termini and in the interstitial regions of many chromosomes not reported previously. A most interesting finding is the presence of 180-bp repeats in the NOR-secondary constriction. TR-1 elements co-exist with 180-bp repeats in the knob on 6S and form alone a small cluster in 4L. 26S and 5S rRNA genes are located in the NOR and at 2L.88, respectively. The combination of chromosome length, centromere position, and distribution of the tandem repeats allows all chromosomes to be identified unambiguously. The results presented form an important basis for using FISH for physical mapping and for investigating genome organization in maize. Received: 29 June 1999 / Accepted: 10 November 1999     

Meiotic checkpoints and the interchromosomal effect on crossing over in Drosophila females

During prophase of meiosis I, genetic recombination is initiated with a Spo11-dependent DNA double-strand break (DSB). Repair of these DSBs can generate crossovers, which become chiasmata and are important for the process of chromosome segregation. To ensure at least one chiasma per homologous pair of chromosomes, the number and distribution of crossovers is regulated. One system contributing to the distribution of crossovers is the pachytene checkpoint, which requires the conserved gene pch2 that encodes an AAA+ATPase family member. Pch2-dependent pachytene checkpoint function causes delays in pachytene progression when there are defects in processes required for crossover formation, such as mutations in DSB-repair genes and when there are defects in the structure of the meiotic chromosome axis. Thus, the pachytene checkpoint appears to monitor events leading up to the generation of crossovers. Interestingly, heterozygous chromosome rearrangements cause Pch2-dependent pachytene delays and as little as two breaks in the continuity of the paired chromosome axes are sufficient to evoke checkpoint activity. These chromosome rearrangements also cause an interchromosomal effect on recombination whereby crossing over is suppressed between the affected chromosomes but is increased between the normal chromosome pairs. We have shown that this phenomenon is also due to pachytene checkpoint activity.     

Integration of the FISH pachytene and genetic maps of Medicago truncatula

A molecular cytogenetic map of Medicago truncatula (2n = 2x = 16) was constructed on the basis of a pachytene DAPI karyogram. Chromosomes at this meiotic prophase stage are 20 times longer than at mitotic metaphase, and display a well differentiated pattern of brightly fluorescing heterochromatin segments. We describe here a pachytene karyogram in which all chromosomes can be identified based on chromosome length, centromere position, heterochromatin patterns, and the positions of three repetitive sequences (5S rDNA, 45S rDNA and the MtR1 tandem repeat), visualized by fluorescence in situ hybridization (FISH). We determined the correlation between genetic linkage groups and chromosomes by FISH mapping of bacterial artificial chromosome (BAC) clones, with two to five BACs per linkage group. In the cytogenetic map, chromosomes were numbered according to their corresponding linkage groups. We determined the relative positions of the 20 BACs and three repetitive sequences on the pachytene chromosomes, and compared the genetic and cytological distances between markers. The mapping resolution was determined in a euchromatic part of chromosome 5 by comparing the cytological distances between FISH signals of clones of a BAC contig with their corresponding physical distance, and showed that resolution in this region is about 60 kb. The establishment of this FISH pachytene karyotype, with a far better mapping resolution and detection sensitivity compared to those in the highly condensed mitotic metaphase complements, has created the basis for the integration of molecular, genetic and cytogenetic maps in M. truncatula.     

Impaired meiotic DNA-damage repair and lack of crossing-over during spermatogenesis in BRCA1 full-length isoform deficient mice

Breast tumor suppressor gene 1 (BRCA1) plays an essential role in maintaining genomic integrity. Here we show that mouse Brca1 is required for DNA-damage repair and crossing-over during spermatogenesis. Male Brca1(Delta11/Delta11)p53(+/-) mice that carried a homozygous deletion of Brca1 exon 11 and a p53 heterozygous mutation had significantly reduced testicular size and no spermatozoa in their seminiferous tubules. During spermatogenesis, homologous chromosomes from the mutant mice synapsed and advanced to the pachytene stage but failed to progress to the diplotene stage. Our analyses revealed that the Brca1 mutation affected cellular localization of several DNA damage-repair proteins. This included prolonged association of gammaH2AX with sites of DNA damage, reduced sex body formation, diminished Rad51 foci and absence of Mlh1 foci in the pachytene stage. Consequently, chromosomes from mutant mice did not form chiasmata, a point that connects exchanging homologous chromosomes. Brca1-mutant spermatocytes also exhibited decreased RNA expression levels of several genes that are involved in DNA-damage repair, including RuvB-like DNA helicase, XPB, p62 and TFIID. Of note, the premature termination of spermatogenesis at the pachytene stage was accompanied by increased apoptosis by both p53-dependent and p53-independent mechanisms. Thus, our study revealed an essential role of Brca1 in DNA-damage repair and crossing-over of homologous chromosomes during spermatogenesis.     

Differential associative behaviour of mitotic and meiotic acrocentric chromosomes

Summary A comparative study of the association of mitotic acrocentric chromosomes and acrocentric bivalents at the pachytene stage shows that at least two factors can act in the associative behaviour of these chromosomes: (1) Nor activity and (2) the presence of satellite DNA in the short arms of these chromosomes. These factors do not act with the same intensity in the two cell lines studied. In lymphocytes, Nor activity prevails, whereas satellite DNA plays the main role in the association of acrocentric chromosomes in germ cells at the pachytene stage.     

Chromosome micro‐dissection and region‐specific libraries from pachytene chromosomes of maize (Zea mays L.)

For the first time, pachytene chromosomes have been used for micro-dissection, amplification and cloning using the short arm of maize chromosome 6. For this purpose, methods for the preparation of unstained pachytene chromosomes were developed. In two separate experiments, 10 and 12 segments of the satellite region were isolated utilizing glass needles and DNA was amplified by linker-adaptor-PCR. After amplification, maize-specific DNA was verified by genomic Southern hybridizations. Chromosomal in situ suppression hybridization confirmed that the adaptor PCR products originated from the micro-dissected 6S region. The adaptor PCR products were cloned into a plasmid vector and the chromosome region-specific libraries were characterized.     

Random acrocentric bivalent associations in human pachytene spermatocytes. Molecular implications in the occurrence of Robertsonian translocations

Acrocentric bivalent associations were studied in 232 human male germ cells at pachytene in order to understand better the preferential involvement of chromosomes 13, 14, and 21 in Robertsonian translocations. The tendency of each acrocentric bivalent to associate with another was not correlated with NOR activity, as measured by silver staining. Good agreement was noticed between their ability to associate and the amount of satellite DNA in human acrocentric chromosomes. The distribution of two-by-two acrocentric bivalent associations was random. In order to reconcile this result with the nonrandom distribution of Robertsonian translocations, a molecular hypothesis is proposed. The model is based on homology of recombinational sites, interspersed at regular interval in satellite DNA, which could increase the probability of accidental unequal crossing-over between two specific acrocentric chromosomes.     

Why plant chromosomes do not show G-bands

Summary Giemsa techniques have refused to reveal G-banding patterns in plant chromosomes. Whatever has been differentially stained so far in plant chromosomes by various techniques represents constitutive heterochromatin (redefined in this paper). Patterns of this type must not be confused with the G-banding patterns of higher vertebrates which reveal an additional chromosome segmentation beyond that due to constitutive heterochromatin. The absence of G-bands in plants is explained as follows: 1) Plant chromosomes in metaphase contain much more DNA than G-banding vertebrate chromosomes of comparable length. At such a high degree of contraction vertebrate chromosomes too would not show G-bands, simply for optical reasons. 2) The striking correspondence of pachytene chromomeres and mitotic G-bands in higher vertebrates suggests that pachytene chromomeres are G-band equivalents, and that this may also be the case in plants. G-banded vertebrate chromosomes are on the average only 2.3 times shorter in mitosis than in pachytene; the chromomeric pattern therefore still can be shown. In contrast, plant chromosomes are approximately 10 times shorter at mitotic metaphase; their pachytene-like arrangement of chromomeres is therefore no longer demonstrable.     

Meiotic checkpoints and the interchromosomal effect on crossing over in Drosophila females

During prophase of meiosis I, genetic recombination is initiated with a Spo11-dependent DNA double-strand break (DSB). Repair of these DSBs can generate crossovers, which become chiasmata and are important for the process of chromosome segregation. To ensure at least one chiasma per homologous pair of chromosomes, the number and distribution of crossovers is regulated. One system contributing to the distribution of crossovers is the pachytene checkpoint, which requires the conserved gene pch2 that encodes an AAA+ATPase family member. Pch2-dependent pachytene checkpoint function causes delays in pachytene progression when there are defects in processes required for crossover formation, such as mutations in DSB-repair genes and when there are defects in the structure of the meiotic chromosome axis. Thus, the pachytene checkpoint appears to monitor events leading up to the generation of crossovers. Interestingly, heterozygous chromosome rearrangements cause Pch2-dependent pachytene delays and as little as two breaks in the continuity of the paired chromosome axes are sufficient to evoke checkpoint activity. These chromosome rearrangements also cause an interchromosomal effect on recombination whereby crossing over is suppressed between the affected chromosomes but is increased between the normal chromosome pairs. We have shown that this phenomenon is also due to pachytene checkpoint activity.