DNase I digestion as a tool for the quantitative evaluation of C-heterochromatic-DNA in situ.

The amount of DNA resisting the C-banding pre-treatments (C-heterochromatic-DNA) was found to account for the interspecific differences of genome size in different Primate groups. The evaluation of this parameter is therefore of great interest in cytotaxonomy. In this work, DNase I digestion was used instead of the pre-treatments C-banding, in an attempt to set up a suitable method for the quantitative evaluation of C-heterochromatic-DNA in both metaphase chromosomes and interphase chromatin. In fact DNase I is known to preferentially digest "active or potentially active" chromatin, and the highly repetitive and inactive DNA in C-heterochromatin should characteristically resist DNase I cleavage. As a model system, differently fixed mouse splenocytes were treated with DNase I for various times, and the digestion was monitored by flow cytometry after propidium iodide staining. In addition, mouse metaphase preparations from lymphocyte cultures were also digested with DNase I, and the amount of residual DNA was evaluated by static microfluorometry. Under controlled conditions of fixation, enzyme concentration, time and temperature, the same limit-digest can be obtained in both interphase nuclei and metaphases, which corresponds to the amount of residual DNA after C-banding and has a C-banding-like pattern in chromosomes.     

Genome size and «C-heterochromatic-DNA» in man and the african apes

The genome sizes and the amounts of DNA after C-banding pretreatments (C-heterochromatic DNA) were measured by quantitative cytochemical methods in man and the African apes,Gorilla gorilla andPan troglodytes. As evaluated by flow cytometry on propidium-iodide-stained lymphocytes, gorilla and chimpanzee have genome sizes larger than man. On the basis of the different resistance of metaphase chromosome DNA to the C-banding procedure, two genome compartments were defined, i.e.,C-heterochromatic-DNA andeuchromatic-DNA. The latter proved to be fairly constant in man and the African apes (as well as in two hylobatid species), whereas the variable amounts ofC-heterochromatic-DNA account well for the interspecific differences of genome size among the hominoid species studied so far. During karyotype diversification, quantitative changes (with either gains or losses) ofC-heterochromatic-DNA seem to have taken place independently in the hylobatid and the man/African ape lineages.     

The AluI-induced bands in great apes and man: implication for heterochromatin characterization and satellite DNA distribution

Restriction endonucleases have recently been proved to be active on fixed chromatin, producing differences in staining of metaphase chromosomes. In this paper we show the results obtained by treating the metaphase chromosomes of Pan troglodytes, Pan paniscus, and Gorilla gorilla with the restriction enzyme AluI. These results demonstrate qualitative differences in the telomeric heterochromatin between Pan and Gorilla despite the fact that these areas appear homogeneous in the two genera by the C-banding method. The results found with individual chromosomes in the different species also appear relevant, in the light of the evolutionary relationships between these nonhuman primates and man. Lastly, the results suggest the presence, in great apes, of some highly repetitive DNA sequences different from the human satellites I-IV.     

5-Methylcytosine in heterochromatic regions of chromosomes: chimpanzee and gorilla compared to the human

Fixed metaphase chromosomes of gorilla and chimpanzee were UV-irradiated to produce regions of single-stranded DNA and then treated with antibodies specific for the minor DNA base 5-methylcytosine (5 MeC). An indirect immunofluorescence technique was used to visualize sites of antibody binding. In the gorilla six pairs of autosomes contained major fluorescent regions, indicating localized regions of highly methylated DNA. These corresponded, with the exception of chromosome 19, to the major regions of constitutive heterochromatin as seen by C-banding. The Y chromosome also contained a highly fluorescent region which was located just proximal to the intense Q-band region. In the chimpanzee no comparable concentrations of highly methylated DNA were seen. Smaller regions of intense 5 MeC binding were present on perhaps six chimpanzee chromosomes, including the Y. Five of these corresponded to chromosomes which were highly methylated in the gorilla.--There is diversity among the human, gorilla and chimpanzee in both the size and location of concentrations of 5 MeC, supporting the idea that satellite DNA evolves more rapidly than DNA in the remainder of the chromosome.     

Modeling heterochromatin regions in transgenic mice

Transgenic mice carrying bovine satellite DNA IV were obtained. The size of the transgene integrated into the mouse genome was approximately 390 kb (about 100 transgene copies) as determined by a semiquantitative PCR. Restriction analysis with isoschizomeric restrictases HpaII and MspI, showed that the alien DNA was methylated. In the genome of a transgenic founder male, two integration sites for satellite DNA IV were revealed by in situ hybridization and in situ PCR. These sites are situated on two different chromosomes: in pericentromeric heterochromatin and within a chromosomal arm. In transgenic mice, de novo formation of heterochromatin regions (C-block and the CMA3 disk within the centromeric heterochromatin of another chromosome) was revealed by C-banding and staining with chromomycin A3. This formation is not characteristic of mice, because their chromosomes normally contain no interstitial C-blocks or sequences intensely stained by chromomycin A3.     

Karyotypes, constitutive heterochromatin, and genomic DNA values in the blowfly genera Chrysomya, Lucilia, and Protophormia (Diptera: Calliphoridae).

The karyotypes and C-banding patterns of Chrysomya species C. marginalis, C. phaonis, C. pinguis, C. saffranea, C. megacephala (New Guinean strain), Lucilia sericata, and Protophormia terraenovae are described. All species are amphogenic and have similar chromosome complements (2n = 12), including an XY-XX sex-chromosome pair varying in size and morphology between species. Additionally, the C-banding pattern of the monogenic species Chrysomya albiceps is presented. The DNA contents of these and of further species Chrysomya rufifacies, Chrysomya varipes, and Chrysomya putoria were assessed on mitotic metaphases by Feulgen cytophotometry. The average 2C DNA value of the male genomes ranged from 1.04 pg in C. varipes to 2.31 pg in C. pinguis. The DNA content of metaphase X chromosomes varied from 0.013 pg (= 1.23% of the total genome) in C. varipes to 0.277 pg (12.20%) in L. sericata; that of Y chromosomes ranged from 0.003 pg (0.27%) in C. varipes to 0.104 pg (5.59%) in L. sericata. In most species, the corresponding 5 large chromosome pairs showed similar relative DNA contents. The data suggest that the interspecific DNA differences in most species are mainly due to quantitative variation of (repetitive) sequences lying outside the centromeric heterochromatin blocks of the large chromosomes. The results are also discussed with regard to phylogenetic relationships of some species.     

Construction of a gorilla fosmid library and its PCR screening system

A gorilla fosmid library of 261,120 independent clones was constructed and characterized. The fosmid vector is similar to the cosmid in average insert size of ca. 40 kb but contains the F factor for replication, and it is more resistant to recombination. This clone library represents about 3.7 times coverage of the gorilla genome. A simple screening system by PCR was established, and we successfully found 9 clones that cover the entire Hox A gene cluster of the gorilla genome. This gorilla fosmid DNA library is a useful resource for comparative genomics of human and apes.     

The characteristics of karyotype and telomeric satellite DNA sequences in the cricket, Gryllus bimaculatus (Orthoptera, Gryllidae)

The chromosomes derived from the Japanese population of Gryllus bimaculatus were characterized by C-banding and Ag-NOR staining. The chromosome number, 2n = 28 + XX (female)/XO (male), corresponded with that of other populations of G. bimaculatus, but the chromosome configuration in idiograms varied between the populations. NORs were carried on one pair of autosomes and appeared polymorphous. The positive C-bands located at the centromere of all chromosomes and the distal regions of many chromosome pairs, and the size and the distribution pattern of the distal C-heterochromatin showed differences among the chromosomes. In addition, this paper reports on the characteristics of HindIII satellite DNA isolated from the genome of G. bimaculatus. The HindIII repetitive fragments were about 0.54 kb long, and localized at the distal C-bands of the autosomes and the interstitial C-bands of the X chromosome. Molecular analysis showed two distinct satellite DNA sequences, named the GBH535 and GBH542 families, with high AT contents of about 67 and 66%, respectively. The two repetitive families seem to be derived from a common ancestral sequence, and both families possessed the same 13-bp palindrome sequence. The results of Southern blot hybridization suggest that the sequence of the GBH535 family is conserved in the genomic DNAs of Gryllus species, whereas the GBH542 family is a species-specific sequence.     

Chromosome banding in Amphibia

Fixed metaphase chromosomes of several species of Amphibia were treated with various restriction endonucleases and subsequently stained with Giemsa. Metaphases of man and chicken were examined in parallel under the same experimental conditions for comparison. The restriction enzymes always induce subsets of the C-banding patterns present in the amphibian karyotypes. The heterochromatic regions can be either resistant or sensitive to the restriction enzyme. The modified C-banding patterns revealed by different restriction endonucleases in the karyotype of the same species can be either extremely dissimilar or almost completely congruent. Correspondingly, the action of the same restriction enzyme on the karyotypes of different species may vary greatly. There is only rarely a correlation between the type of C-banding patterns produced by different restriction endonucleases and their specific base pair recognition sequences. In contrast to mammalian and avian chromosomes, restriction enzymes induce no multiple G-banding patterns in amphibian chromosomes. This is attributed to the difference in organization of the DNA in the genomes of poikilothermic vertebrates. The possible mechanisms of restriction endonuclease banding and the various uses of this technique for amphibian chromosomes are discussed.     

Satellite DNA specific to knob heterochromatin inCucumis metuliferus (Cucurbitaceae)

Buoyant density gradient analysis of nuclear DNA of fourCucumis species showed asymmetric profiles indicating the presence of satellite DNA sequences in the nuclear genome. A highly repeated satellite DNA sequence was isolated from the nuclear genome ofC. metuliferus under neutral CsCl gradients. The satellite DNA constitutes about 4.96% of total nuclear DNA and has 48.06% guanine plus cytosine content. The kinetic complexity of satellite DNA is 150 times smaller than T₄ phage DNA and the base sequence divergence is low.³H-labeled cRNA transcribed from satellite DNA hybridized clearly to six heterochromatic knobs of pachytene chromosomes. The knob heterochromatin can be distinguished by Giemsa C-banding of pachytene chromosomes. Restriction enzyme analysis and Southern blot hybridization indicated that the satellite DNA has a tandem arrangement and predominantly formed two bands of size 210 and 151 base pairs. Absence of knob satellite DNA ofC. metuliferus in the nuclear genomes ofC. melo, C. anguria andC. sativus showed thatC. metuliferus remains isolated within the genusCucumis.     

Influence of the C-banding procedure on DNA and proteins of mouse chromosomes: I. A microdensitometric study

Cytochemical quantitative methods were used to investigate DNA protein contents of mouse metaphase plates during an alkaline C-banding procedure ( Sumner et al., 1971). Cytochemical stains and reactions for DNA and for total protein content were used to quantitatively assess the sequential involvement (losses) of DNA and protein during the appearance of the classic C-banding pattern which was monitored with Giemsa staining. The data point the preferential loss of DNA from euchromatic regions of chromosomes as the main cause of the C-banding pattern appearance. The effect of chromosomal protein is more likely indirect and perhaps tied to some specific interaction with centromeric DNA that contributes to DNA retention in C-bands. Following the C-banding procedure it was possible to differentially stain the centromeric area with Feulgen and GCA and even with non-fully specific stain for DNA such as methylene blue.     

C-banded karyotypes of two Silene species with heteromorphic sex chromosomes.

Mitotic metaphase chromosomes of Silene latifolia (white campion) and Silene dioica (red campion) were studied and no substantial differences between the conventional karyotypes of these two species were detected. The classification of chromosomes into three distinct groups proposed for S. latifolia by Ciupercescu and colleagues was considered and discussed. Additionally, a new small satellite on the shorter arm of homobrachial chromosome 5 was found. Giemsa C-banded chromosomes of the two analysed species show many fixed and polymorphic heterochromatic bands, mainly distally and centromerically located. Our C-banding studies provided an opportunity to better characterize the sex chromosomes and some autosome types, and to detect differences between the two Silene karyotypes. It was shown that S. latifolia possesses a larger amount of polymorphic heterochromatin, especially of the centromeric type. The two Silene sex chromosomes are easily distinguishable not only by length or DNA amount differences but also by their Giemsa C-banding patterns. All Y chromosomes invariably show only one distally located band, and no other fixed or polymorphic bands on this chromosome were observed in either species. The X chromosomes possess two terminally located fixed bands, and some S. latifolia X chromosomes also have an extra-centric segment of variable length. The heterochromatin amount and distribution revealed by our Giemsa C-banding studies provide a clue to the problem of sex chromosome and karyotype evolution in these two closely related dioecious Silene species.     

Climate and growth form: the consequences for genome size in plants

The adaptive significance of nuclear DNA variation in angiosperms is still widely debated. The discussion mainly revolves round the causative factors influencing genome size and the adaptive consequences to an organism according to its growth form and environmental conditions. Nuclear DNA values are now known for 3874 angiosperm species (including 773 woody species) from over 219 families (out of a total of 500) and 181 species of woody gymnosperms, representing all the families. Therefore, comparisons have been made on not only angiosperms, taken as a whole, but also on the subsets of data based on taxonomic groups, growth forms, and environment. Nuclear DNA amounts in woody angiosperms are restricted to less than 23.54 % of the total range of herbaceous angiosperms; this range is further reduced to 6.8 % when woody and herbaceous species of temperate angiosperms are compared. Similarly, the tropical woody dicots are restricted to less than 50.5 % of the total range of tropical herbaceous dicots, while temperate woody dicots are restricted to less than 10.96 % of the total range of temperate herbaceous dicots. In the family Fabaceae woody species account for less than 14.1 % of herbaceous species. Therefore, in the total angiosperm sample and in subsets of data, woody growth form is characterized by a smaller genome size compared with the herbaceous growth form. Comparisons between angiosperm species growing in tropical and temperate regions show highly significant differences in DNA amount and genome size in the total angiosperm sample. However, when only herbaceous angiosperms were considered, significant differences were obtained in DNA amount, while genome size showed a non-significant difference. An atypical result was obtained in the case of woody angiosperms where mean DNA amount of tropical species was almost 25.04 % higher than that of temperate species, which is because of the inclusion of 85 species of woody monocots in the tropical sample. The difference becomes insignificant when genome size is compared. Comparison of tropical and temperate species among dicots and monocots and herbaceous monocots taken separately showed significant differences both in DNA amount and genome size. In herbaceous dicots, while DNA amount showed significant differences the genome size varies insignificantly. There was a non-significant difference among tropical and temperate woody dicots. In three families, i.e., Poaceae, Asteraceae, and Fabaceae the temperate species have significantly higher DNA amount and genome size than the tropical ones. Woody gymnosperms had significantly more DNA amount and genome size than woody angiosperms, woody eudicots, and woody monocots. Woody monocots also had significantly more DNA amount and genome size than woody eudicots. Lastly, there was no significant difference between deciduous and evergreen hardwoods. The significance of these results in relation to present knowledge on the evolution of genome size is discussed.     

Development of Triticum aestivum-Leymus racemosus translocation lines using gametocidal chromosomes

Maan[1] and Endo[2] et al. first reported that some chromosomes from Ae. longgissima, Ae. sharonensis and Ae. triuncialis showed preferential transmission when introduced into wheat background. The mechanism for this phenomenon rests with the fact that contrary to the normal fertility of gametes with these chromosomes, chromosome structural aberrations occur seriously in the gametes without these chromosomes, causing less compatibility in selective fertilization and resulting in semi-sterilit…     

Physical Mapping of Rice Chromosomes 8 and 9 with YAC Clones

First efforts for physical mapping of rice chromosomes 8 and9 were carried out by ordering YAC clones of a rice genomicDNA library covering six genome equivalents with mapped DNAmarkers. A total of 79 and 74 markers from chromosomes 8 and9, respectively, were analyzed by YAC colony and Southern hybridizationusing RFLP markers of cDNA and genomic clones, and by polymerasechain reaction (PCR) screening using PCR-derived and sequence-taggedsite (STS) markers. As a result, 252 YAC clones were confirmedto contain the mapped DNA fragments on both chromosomes. A contigmap was constructed by ordering these YAC clones and about 53%and 43% genome coverage was obtained for chromosomes 8 and 9,respectively, assuming a YAC clone size of 350 kb and overlapbetween neighboring YACs of 50%. A continuous array of YAC cloneswith minimum overlap gave a total size of 18.9 Mb for chromosome8 and 15.6 Mb for chromosome 9, which are close to previousestimates. These contig maps may provide valuable informationthat can be useful in understanding chromosome structure andisolating specific genes by map-based cloning.     

Comparison of the chromosomes of Triticum timopheevi with related wheats using the techniques of C-banding and in situ hybridization

Summary The chromosomes of the tetraploid wheats Triticum timopheevi (Genome AAGG) and T. araraticum (Genome AAGG) were C-banded at mitosis. The identity of the banded and unbanded chromosomes was then established by firstly making comparisons with the hexaploid species T. zhukovskyi which has the genome formula AAAAGG. Secondly, the meiotic pairing in F₁ hybrids between T. timopheevi and diploid wheats was examined by means of C-banding. The results showed that the banded chromosomes belonged to the G genome, while the unbanded chromosomes belonged to the A genome. Only one of the two pairs of satellited chromosomes had strong heterochromatic bands. The relationship between the genomes of T. timopheevi and T. dicoccum (Genome AABB) was then assessed at meiosis in hybrids between these species, using the techniques of C-banding and in situ hybridisation of a cloned ribosomal RNA gene probe. It was concluded that there were differences both in the amount and distribution of heterochromatin and also translocation differences between the species.     

The distribution of mitomycin C-induced sister chromatid exchanges in the euchromatin and heterochromatin of the Indian Muntjac

The frequency of sister chromatid exchanges (SCEs) induced by mitomycin C (MMC) in Indian Muntjac chromosomes was determined by the fluorescence plus Giemsa (FPG) technique. Using scanning cytophotometry the relative DNA content of each chromosome was measured with and without acid or alkali pretreatments for C-banding. During acid and alkali treatments, euchromatin lost 20 to 30% of its DNA, while heterochromatin lost less than 5%; an intermediate DNA loss was observed for the short arm of the X chromosome. After growth of cells in the presence of MMC during the first cycle and in the presence of bromodeoxyuridine (BrdU) during the first and second cycles of DNA replication, SCEs in the euchromatin were proportional to DNA content. SCEs at the junctions between the neck of the X chromosome and the long and short arms occurred more frequently than expected. A threshold effect for the induction of SCEs was observed in regions resistant to DNA extraction by acid and alkali treatments (i.e., the neck and short arm of the X chromosome). At high concentrations of MMC, the frequency of SCE at each junction appears to plateau at 0.5.     

Banding Human Chromosomes Using a Combined C-Banding-Fluorochrome Staining Technique

Slides pretreated for C-banding and stained with DAPI or CMA3 show different banding patterns in human metaphase chromosomes compared to those obtained with either standard Giemsa C-banding or fluorochrome staining alone. Human chromosomes show C-plus DA-DAPI banding after C-banding plus DAPI and enhanced R-banding after C-banding plus Chromomycin A3 staining. If C-banding preferentially removes certain classes of DNA and proteins from different chromosome domains, C-banding pre-treatment may cause a differential DNA extraction from G- and R-bands in human chromosomes, resulting in a preferential extraction of DNA included in G-bands. This hypothesis is partially supported by the selective cleavage and removal of DNA from R-bands of restriction endonuclease HaeIII with C-banding combined with DAPI or Chromomycin A3 staining. Structural factors relating to regional differences in DNA and/or proteins could also explain these results.     

Evaluation of phylogenetic relationships between five polyploid Aegilops L. species of the U-genome cluster by means of chromosomal analysis

Four tetraploid (Aegilops ovata, Ae. biuncialis, Ae. columnaris, and Ae. triaristata) and one hexaploid (Ae. recta) species of the U-genome cluster were studied using C-banding technique. All species displayed broad C-banding polymorphism and high frequency of chromosomal rearrangements. Chromosomal rearrangements were represented by paracentric inversions and intragenomic and intergenomic translocations. We found that the processes of intraspecific divergence of Ae. ovata, Ae. biuncialis, and Ae. columnaris were probably associated with introgression of genetic material from other species. The results obtained confirmed that tetraploid species Ae. ovata and Ae. biuncialis occurred as a result of hybridization of a diploid Ae. umbellulata with Ae. comosa and Ae. heldreichii, respectively. The dissimilarity of the C-banding patterns of several chromosomes of these tetraploid species and their ancestral diploid forms indicated that chromosomal aberrations might have taken place during their speciation. Significant differences of karyotype structure, total amount and distribution of C-heterochromatin found between Ae. columnaris and Ae. triaristata, on the one hand, and Ae. ovata and Ae. biuncialis, on the other, evidenced in favor of different origin of these groups of species. In turn, similarity of the C-banding patterns of Ae. columnaris and Ae. triaristata chromosomes suggested that they were derived from a common ancestor. A diploid species Ae. umbellulata was the U-genome donor of Ae. columnaris and Ae. triaristata; however, the donor of the second genome of these species was not determined. We assumed that these tetraploid species occurred as a result of introgressive hybridization. Similarity of the C-banding patterns of chromosomes of Ae. recta and its parental species Ae. triaristata and Ae. uniaristata indicated that the formation of the hexaploid form was not associated with large modifications of the parental genomes.     

Electrophoretic karyotyping of the lignin-degrading basidiomycete Phanerochaete chrysosporium

Electrophoretic karyotyping of the two most widely studied strains of Phanerochaete chrysosporium, BKMF-1767 and ME-446, has been determined using transverse alternating field etectrophoresis. The genomic DNA of BKMF-1767 was resolved into 10 chromosomes ranging in size from 1.8–5.0 Mb, amounting to a total genome size of about 29 Mb. The genomic DNA of strain ME-446, on the other hand, was resolved into 11 chromosomes, amounting to a total genome size of about 32Mb. Lignin peroxidase genes have been localized to five chromosomes in strain BKMF-1767 and to four chromosomes in strain ME-446.