Chromosome Science Vol. 19(2016) No. 1-4

The silkworm, Bombyx mori, is a lepidopteran model with long history of the domesticated insect for silk production, which contributed to human life as well as insect sciences. In chromosome science, the silkworm could be the first record of karyotype count in Lepidoptera. Because of the holokinetic chromosomes, precise chromosome identification and karyotype had been difficult until BAC-FISH (fluorescence in situ hybridization with bacterial chromosome (BAC) DNAs as probes) was applied for the silkworm chromosome analysis. Here we review the research histories for the first B. mori karyotype and its contribution for chromosome science and comparative genomics in Lepidoptera.

Chromosome elimination and chromatin diminution occur in various species including single-cell ciliates and several multicellular animals. DNA methylcytosine (5mC) and histone modifications have been identified as markers of the eliminated DNA and chromatins in ciliate and finch. Here we examined the levels of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC; an intermediate of active DNA demethylation) in the male testicular cells of the inshore hagfish Eptatretus burgeri and simultaneously detected germline-restricted repetitive sequences (EEEb1) to identify the chromosomes restricted to germ cells (E-chromosomes). We detected 5mC and 5hmC signals at all chromosomes in the spermatogonia and in all of the interphase nuclei whereas 5mC signals were selectively located on the chromosomes without EEEb1 signals in the spermatocytes' metaphase, suggesting no 5mC signal on the E-chromosomes. No significant difference in 5hmC levels between the E-chromosomes and the other chromosomes, was detected in the spermatocytes. This chromosome-specific hypomethylation has never been detected in mouse or zebrafish germ cells. These results therefore suggest that the DNA methylation pattern of the E-chromosomes, namely those presumptively eliminated in somatic differentiation, are altered just before or during meiosis. This exclusive alteration of the methylation pattern may play a key role in the chromosome elimination in hagfish species' embryogenesis.

The structural details of chromosomes have been of interest for many years; however, the enigma of how the metaphase chromosome is constructed has remained unsolved. Divalent cations, especially Mg²⁺, are known to be required for chromatin condensation. However, details about the effect of Mg²⁺ at the nanoscale are still limited. In this study, the effect of Mg²⁺ on chromosome structure was investigated by means of scanning electron microscopy (SEM), because of its high magnification and resolution, as well as by scanning transmission electron microscope (STEM) tomography, because of its advantages in three-dimensionally imaging chromosomes without sectioning. We herewith report the reversibility between 11 and 30 nm chromosome structures according to the concentration of Mg²⁺ as observed by SEM, and how three-dimensional chromosome structure is affected by Mg²⁺ concentration by STEM tomography. Treatment with a buffer lacking Mg²⁺ yields a less compact chromosome structure, with smaller fiber diameters, than for chromosomes treated with a buffer containing 5 mM Mg²⁺. The changes of chromatin diameter are reversible after re-addition of Mg²⁺. These findings signify the importance of an adequate concentration of Mg²⁺ to chromosome structure. The advantages of SEM and STEM for chromosomal research were highlighted in the current study.

Despite the efforts of numerous researchers over the years, inner structure of a chromosome is still controversial, although several models have been proposed to date. It is now well known that there are two important structural components to the chromosome, the chromosome scaffold and chromatin fibers. The chromosome scaffold, which is mainly composed of four different proteins, is a protein axis extending longitudinally in both chromatids. The chromatin fiber, which is composed of a DNA strand with histone proteins, is also packed in each chromatid. We used focused ion beam/scanning electron microscope (FIB/SEM) to elucidate these two structural components using human chromosomes. FIB/SEM effectively cuts the human chromosomes by its focused Ga ion beam and the cross-sections were visualized by the resolution of scanning electron microscope. As a result, the chromosome scaffold has been confirmed to be located in the central region of each chromatid. The distribution of chromatin fiber in the chromosome's inner space was also detected. It seems to be distributed more or less randomly within a chromosome. These results strongly indicated that the nanotechnology afforded by FIB/SEM is an effective method to reveal the chromosome's inner structure in detail.

The locations of the 18S-5.8S-25S and 5S ribosomal RNA genes (rDNAs) on the chromosomes in the seedlings obtained from open-pollination of apple ‘Sensyu’ (Malus × domestica Borkh.) were determined using fluorescence in situ hybridization (FISH). 18S-5.8S-25S and 5S rDNA probes were labeled with biotin and hybridization signals were detected using a fluorescein isothiocyanate (FITC)-avidin conjugate on the chromosomes counterstained with DAPI. The 18S-5.8S-25S rDNA signals were detected in the telomeric positions of eight chromosomes among 34. These sites were located on two long, two relatively long, two medium, and two relatively short chromosomes. The two 5S rDNA sites were located at centromeric positions of relatively short chromosomes which do not possess 18S-5.8S-25S rDNA sites. The numbers and positions of rDNA sites were stable among the seedlings.

Although Torenia fournieri (2n=2x=18) and Torenia baillonii (2n=2x=16) have different chromosome numbers, almost all of the parental chromosomes form bivalents by interspecific pairing during meiosis in interspecific hybrids. Here, we produced another hybrid between the two species and its six BC₁F₁ progenies (F₁ hybrid × T. baillonii). These plants had previously unreported chromosome compositions: the total chromosome number was 34, as expected for allotetraploids, but some T. fournieri chromosomes were gained and some T. baillonii chromosomes were lost. Plants with these new karyotypes grew well and showed different morphologies. This study indicates that two parental genomes in interspecific hybrids share several interchangeable homoeologous chromosomes.

Across species, eukaryotic chromosomes share common features at the molecular level. However, common features at the cellular level are not well investigated. A correlation has been suggested between the linear packing ratio of mitotic chromosomes and the intra-nuclear DNA density, by comparing these values in the nematode Caenorhabditis elegans. In this study, these values were measured and compared among several metazoan and plant species. The obtained values corroborated the correlation proposed in the previous study, supporting the theory that intra-nuclear DNA density is a common regulator of chromosome condensation. Moreover, the comparison among different species suggested a correlation between the length of a mitotic chromosome and the nuclear volume to the power of 2/3. Given this observation, we speculate that: (i) a rate-limiting component defines the length of a mitotic chromosome that is proportional to the nuclear surface area, and (ii) such regulation of the mitotic chromosomal length may play a role in maintaining the ratio between the cell size and the metaphase plate.

Following its recent rapid development, nanotechnology is now an effective tool in chromosome science and technology (Fukui and Ushiki 2007). It can be used effectively for both functional and structural studies of chromosomes, although its direct advantages have been shown mainly in structural studies. Thus, it is expected that nanotechnology will be extensively used in the field of chromosome structure. This review describes three of the most promising and effective nanotechnologies and related technology: super-resolution microscopy (in particular, three-dimensional structured illumination microscopy, 3D-SIM), focused ion beam/scanning electron microscopy (FIB/SEM), and scanning transmission electron microscopy (STEM). Their applications in the elucidation of higher-order chromosome structure are presented and discussed based on the achievements already attained.