An easy protocol for studying chromatin and recombination protein dynamics during Arabidopsis thaliana meiosis: immunodetection of cohesins, histones and MLH1.

Plant meiosis studies have enjoyed a fantastic boom in recent years with the use of Arabidopsis thaliana as a model not only for molecular genetics and genomics but also for cytogenetics. In this article we describe a new protocol for immunolabelling meiotic proteins that allows the detection of a large range of proteins on strongly spread chromosomes throughout the entire meiotic process. We used this method to immunodetect MLH1, a crucial component of the meiotic recombination machinery, and found that it can be visualised as foci from pachytene to diakinesis, where it co-localises with chiasmata. The mean MLH1 foci number per meiotic cell at diakinesis was 8.4 for WS-4 and 9.95 for Col-0, with the number of foci per bivalent ranging from 1 to 5. We also analysed MLH1 distribution within bivalents and found that they were not restricted to specific chromosomal regions. The analysis of MLH1 foci formation in the Atzip4 mutant, where class I crossover (CO) formation is prevented, revealed that residual chiasmata were not labelled by MLH1, strongly suggesting that MLH1 antibodies only label class I COs in Arabidopsis. It thus appears that the 'obligatory CO' is systematically labeled by MLH1 and is generated through the class I pathway.

Chromosome 'speed dating' during meiosis of polyploid Brassica hybrids and haploids.

Given their tremendous importance for correct chromosome segregation, the number and distribution of crossovers are tightly controlled during meiosis. In this review, we give an overview of crossover formation in polyploid Brassica hybrids and haploids that illustrates or underscores several aspects of crossover control. We first demonstrate that multiple targets for crossover formation (i.e. different but related chromosomes or duplicated regions) are sorted out during meiosis based on their level of relatedness. In euploid Brassica napus (AACC; 2n = 38), crossovers essentially occur between homologous chromosomes and only a few of them form between homeologues. The situation is different in B. napus haploids in which crossovers preferentially occur between homeologous chromosomes and a few can then form between more divergent duplicated regions. We then provide evidence that the frequency of crossovers between a given pair of chromosomes is influenced by the karyotypic and genetic composition of the plants that undergo meiosis. For instance, genetic evidence indicates that the number of crossovers between exactly the same pairs of homologous A chromosomes gets a boost in Brassica digenomic tetraploid (AACC) and triploid (AAC) hybrids. Increased autosyndesis within B. napus haploids as compared to monoploid B. rapa and B. oleracea is another illustration of this process. All these observations may suggest that polyploidization overall boosts up crossover machinery and/or that the number of crossovers is modulated through inter-bivalents or univalent-bivalent cross-talk effects. The last part of this review gives an up-to-date account of what we know about the genetic control of homologous and homeologous crossover formation among Brassica species.

Pairing and recombination at meiosis of Brassica rapa (AA) x Brassica napus (AACC) hybrids.

Interspecific crosses contribute significantly to plant evolution enabling gene exchanges between species. The efficiency of interspecific crosses depends on the similarity between the implicated genomes as high levels of genome similarity are required to ensure appropriate chromosome pairing and genetic recombination. Brassica napus (AACC) is an allopolyploid, resulting from natural hybridization between Brassica rapa (AA) and Brassica oleracea (CC), both being diploid species derived from a common ancestor. To study the relationships between genomes of these Brassica species, we have determined simultaneously the pairing and recombination pattern of A and C chromosomes during meiosis of AAC triploid hybrids, which result from the interspecific cross between natural B. napus and B. rapa. Different AAC triploid hybrids and their progenies have been analysed using cytogenetic, BAC-FISH, and molecular techniques. In 71% of the pollen mother cells, homologous A chromosomes paired regularly, and usually one chromosome of each pair was transmitted to the progeny. C chromosomes remained mainly univalent, but were involved in homoeologous pairing in 21.5% of the cells, and 13% of the transmitted C chromosomes were either recombined or broken. The rate of transmission of C chromosomes depended on the identity of the particular chromosome and on the way the hybrid was crossed, as the male or as the female parent, to B. napus or to B. rapa. Gene transfers in triploid hybrids are favoured between A genomes of B. rapa and B. napus, but also occur between A and C genomes though at lower rates.

[The lampbrush chromosomes of the chicken. Cytological maps of the macrobivalents]. / Khromosomy-lampovye shchetki kuritsy. Tsitologicheskie karty makrobivalentov.

The lampbrush chromosomes (LBC) were prepared from growing oocytes 0.75-1.50 mm in diameter. A map of 6 autosomes and the ZW sex bivalents is presented. Several types of landmarks were noticed: lumpy loops (LL), telomeric bow-like loops (TBL), some large loops in interstitial regions (marker loops--ML). Supposedly, the centromeres of LBC in the chicken are at one of the axial bars bearing no loops. The landmarks PBL and DBL mark the proximal and distal boundaries of bars. LBC-A (probably, chromosome 1 of the chicken karyotype) is about 185 microns. There are 7.3 +/- 0.2 chiasmata. Chiasmata are distributed at quasi-random. In LBC-A one chiasma is localized in a telomere, as a rule. Coordinates of 13 of the 14 different landmarks in LBC-A have been estimated. LBC-B (probably, chromosome 2) is about 151 microns, there are 5.50 +/- 0.23 chiasmata. The LBC-B may be identified by LL-21 and LL-22. LBC-C (probably, chromosome 3) is 128 microns; there are 4.70 +/- 0.18 chiasmata. The chromosome can be identified by characteristic loops LL-31, an unlooped chromomere bar near the telomere (T-32), a characteristic distribution of normal loops along LBC-C: about one half of this LBC bears large loops, and the other one--small loops. LBC-D (chromosome 4?) is 107 microns; there are 3.80 +/- 0.31 chiasmata. Double-loop bridges appear frequently near ML-41. LBC-E (chromosome 5?) is about 72 microns with 2.50 +/- 0.28 chiasmata. There are characteristic TBL loops with abundant RNP material thus being like LL-loops. LBC-F (chromosome 8?) is about 36.5 microns; there are 2 chiasmata. This LBC can be identified by giant telomeric loops GML-F1 and by unlooped bar in the middle of LBC.

[Heterochromatin regions of the chromosomes of the hen and Japanese quail in mitosis and at the lampbrush stage]. / Geterokhromatinovye raiony khromosom kuritsy i iaponskogo perepela v mitoze i na stadii lampovykh shchetok.

The mitotic and lampbrush chromosomes of the domestic fowl and Japanese quail were analysed by fluorochrome staining technique. The lampbrush chromosomes of both the subjects displayed a typical "loop-chromomere" structure. Three distinct kinds of loops were distinguished in Gallus g. domesticus--normal, telomeric bows, and lumps. The former are distributed along the whole chromosome length. The latter and the bows were observed in subtelomeric and telomeric regions. By DNA/RNA specific acridine orange staining it was shown that each loop (especially, "lumpy" loops) contained a rich RNP matrix. A comparative analysis of the chromomycin A3/distamycin A banding pattern of mitotic and lampbrush chromosomes shows that the telomeric "bows" and "lumps" are special loops developed in telomeric heterochromatic bands. In Coturnix c. japonica, the CMA/DA-positive bands were not observed in telomeres of mitotic macrochromosomes, except a smallest band in the 2p-arm telomere. The absence of telomeric heterochromatic bands which can be visualized in the quail mitotic chromosomes coincides with the absence of "bow"-like loops. Only small lump-like structures were seen in some telomeres of macroautosomes. The biological significance of loop formation and RNA synthesis in heterochromatic band loops in growing oocytes is briefly discussed.

[Molecular and cytologic analyses of the transcription of evolutionarily conservative moderately repetitive sequences of vertebrate genomes]. / Molekuliarnyi i tsitologicheskii analiz transkriptsii évoliutsionno konservativnykh umerenno povtoriaiushchikhsia posledovatel'nostei genomov pozvonochnykh.

Transcription of several families of moderately repeated sequences, conserved through the evolution of vertebrates, has been studied in different types of pigeon, chicken and mouse cells. It is shown both by hybridization with isolated RNA and by in situ hybridization that the families of repeats, dispersed in bird genomes and organized in clusters, are differentially expressed in pigeon erythroid cells with different degrees of specialization; in addition, they are transcribed in different types of chick embryo cells and on lampbrush chromosomes in chicken oocytes. Sequences homologous to these repeats were transcribed in different types of newborn mouse cells. Another family of conservative moderate repeats (family T1) dispersed in the mouse genome was also transcribed in a large variety of tissues in both new-born mice and chick embryos. A comparison of structural and transcription features of conservative moderate repeats represented in genomes of the number of vertebrates made it possible to regard them as "housekeeping" elements. The conservation in the evolution as well as the character of transcription of similar genome elements testify to their important role in the organism functioning at different stages of development.

[Molecular and cytologic analysis of clusters of moderate repeats of bird genomes]. / Molekuliarnyi i tsitologicheskii analiz klasterov umerennykh povtorov genomov ptits.

Pigeon genome long sequences containing clusters of moderately repeating elements have been cloned. Molecular analysis has shown a dispersed distribution of the repeats in both pigeon and chicken genomes. Within a single cluster, a scrambled distribution of elements belonging to different families of repeats has been shown. Similar repeated sequences have been revealed within clusters. The analysed clusters of repeats are characterized by a limited structural variability in the genomes. In situ hybridization revealed the localization of sequences complementary to the cloned clusters in pigeon and chicken macrochromosomes. Preferential localization has been demonstrated in telomeric and centromeric chromosome regions as well as in the region of R-bands.