Location of repetitious DNA in the chromosomes of the desert locust (Schistocerca gregaria)

A modified Giemsa staining technique and the in situ hybridisation technique, have been used to investigate the localisation of highly repeated sequences in the karyotype of the locust Schistocerca gregaria. The centromeric regions are stained densely with Giemsa and further Giemsa-stained bands occur at the telomeric region of the short (S) chromosomes. RNA complementary to repetitious DNA hybridised to loci scattered along the whole length of the chromosomes, with concentrations of grains at the centromeric regions of all the chromosomes and also at the telomeric regions of the S chromosomes.     

Giemsa banding of meiotic chromosomes

Applying a Giemsa staining technique to the meiotic chromosomes of Anemone blanda demonstrates that Giemsa bands similar to those seen in the mitotic chromosomes are discernible at all the principal stages of meiosis. The bands are not a product of the Giemsa procedure since they can be seen in unstained preparations using phase-contrast optics as chromocentres in interphase nuclei and as condensed regions in prophase chromosomes. That the bands seem to be permanent features of the nucleus, whether it is dividing or otherwise is an important consideration for understanding their nature and function. Bands and chiasmata do not coincide indicating on the one hand that chiasmata are not responsible for differences in banding patterns and on the other hand that the conservation of bands is an indication that they are either inert regions or specialised regions with considerable adaptive significance. These alternatives can only be resolved by genetical studies of the banding phenomena.     

The organization of DNA in the mitotic and polytene chromosomes of Sciara coprophila

The organization of DNA in the mitotic metaphase and polytene chromosomes of the fungus gnat, Sciara coprophila, has been studied using base-specific DNA ligands, including anti-nucleoside antibodies. The DNA of metaphase and polytene chromosomes reacts with AT-specific probes (quinacrine, DAPI, Hoechst 33258 and anti-adenosine) and to a somewhat lesser extent with GC-specific probes (mithramycin, chromomycin A₃ and anticytidine). In virtually every band of the polytene chromosomes chromomycin A₃ fluorescence is almost totally quenched by counterstaining with the AT-specific ligand methyl green. This indicates that GC base pairs in most bands are closely interspersed with AT base pairs. The only exceptions are band IV-8A3 and the nucleolus organizer on the X. In contrast, quinacrine and DAPI fluorescence in every band is only slightly quenched by counterstaining with the GC-specific ligand actinomycin D. Thus, each band contains a moderate proportion of AT-rich DNA sequences with few interspersed GC base pairs. — The C-bands in mitotic and polytene chromosomes can be visualized by Giemsa staining after differential extraction of DNA and those in polytene chromosomes by the use of base-specific fluorochromes or antibodies without prior extraction of DNA. C-bands are located in the centromeric region of every chromosome, and the telomeric region of some. The C-bands in the polytene chromosomes contain AT-rich DNA sequences without closely interspered GC base pairs and lack relatively GC-rich sequences. However, one C-band in the centromeric region of chromosome IV contains relatively GC-rich sequences with closely interspersed AT base pairs. — C-bands make up less than 1% of polytene chromosomes compared to nearly 20% of mitotic metaphase chromosomes. The C-bands in polytene chromosomes are detectable with AT-specific or GC-specific probes while those in metaphase chromosomes are not. Thus, during polytenization there is selective replication of highly AT-rich and relatively GC-rich sequences and underreplication of the remainder of the DNA sequences in the constitutive heterochromatin.     

Chromatin differentiation between Theobroma cacao L. and T. grandiflorum Schum

A comparative analysis of mitotic chromosomes of Theobroma cacao (cacao) and T. grandiflorum (cupuaçu) was performed aiming to identify cytological differences between the two most important species of this genus. Both species have symmetric karyotypes, with 2n = 20 metacentric chromosomes ranging in size from 2.00 to 1.19 μm (cacao) and from 2.21 to 1.15 μm (cupuaçu). The interphase nuclei of both species were of the arreticulate type, displaying up to 20 chromocentres, which were more regularly shaped in cacao than in cupuaçu. Prophase chromosomes of both species were more condensed in the proximal region, sometimes including the whole short arm. Both species exhibited only one pair of terminal heterochromatic bands, positively stained with chromomycin A ₃ , which co-localized with the single 45S rDNA site. Each karyotype displayed a single 5S rDNA site in the proximal region of another chromosome pair. Heterochromatic bands were also observed on the centromeric/pericentromeric regions of all 20 chromosomes of cacao after C-banding followed by Giemsa or DAPI staining, whereas in cupuaçu they were never detected. These data suggest that the chromosomes of both species have been largely conserved and their pericentromeric chromatin is the only citologically differentiated region.     

The behaviour and structure of the sex chromosomes in spermatocytes of Microtus oeconomus

The meiotic behaviour and structure of the sex chromosomes of Microtus oeconomus (2n=30) in Giemsa stained preparations are described. The X-Y pair appears as a sex vesicle at late zygotene. At late pachytene an unfolded sex vesicle is visible. A condensed sex vesicle appears during pre-diffuse diplotene and starts to unfold again during post-diffuse diplotene. At diakinesis and metaphase I the X and Y chromosomes can be recognized in an end-to-end association. During anaphase I, interkinesis and metaphase II the sex chromosomes are heteropycnotic and can therefore easily be recognized during the final stages of meiosis. During spermiogenesis the X and Y chromosomes can be identified in Giemsa stained preparations until the stage of spermatid elongation.     

The role of chromosomal proteins in the C-banding of Allium cepa chromosomes.

When chromosomes of Allium cepa are subjected to a C-banding procedure (incubation in saturated barium hydroxide followed by phosphate buffer at 60 degrees C for 1 h) and then treated with Giemsa stain, bands appear at the telomeres of all chromosomes. Microspectrophotometric measurements of Feulgen-DNA content, demonstrated that the C-banding procedure extracted DNA from the nuclei. Staining of banded chromosomes with several DNA-specific stains showed that this loss was differential, with the band DNA exhibiting more resistance to extraction than that of the rest of the chromosome. The C-banding procedure did not extract chromosomal proteins, however, and no difference in mass per unit length could be detected by Nomarski optics between band and interband regions. Several experiments demonstrated that chromosomal proteins play a significant role in C-banding. First, treatment of chromosomes with pronase before C-banding resulted in the elimination of differential staining with Giemsa. Furthermore, in preparations where the DNA was completely hydrolysed with hot TCA, the remaining chromosomal proteins were found to exhibit a differential affinity for Giemsa stain. Amido black staining demonstrated that total chromosomal protein was uniformly distributed after the hot TCA digestion, but the proteins localized in the telomeres had a greater affinity for the Giemsa stain than the bulk of the chromosomal proteins. When the TCA-digested chromosomes were subjected to the C-banding procedure before staining, the differential affinity of the telomeres for the Giemsa stain was lost. Thus, C-banding appears to be the result of a complex interaction between protein and DNA in which the greater resistance to extraction of the band DNA is necessary to stabilize and preserve chromatin protein which exhibits a differential affinity for Giemsa stain.     

The karyotype of the golden monkey (Rhinopithecus r. roxellanae)

In this paper, the karyotype and G-banding pattern of the chromosomes of cultured peripheral blood lymphocytes in R. r. roxellanae were investigated. The chromosome number of this species is 44 in both sexes. In R. r. roxellanae, as in other monkeys, sex is determined by specific sex chromosomes, i.e. the male is XY and the female is XX. The 21 pairs of autosomes consist of 7 pairs of metacentric chromoomes, 13 pairs of submetacentric chromosomes and one acrocentric pair. Chromosome measurements were made from highly enlarged photographic prints. They included the relative length, arm ratio and centromere index of each chromosome. Both chromosomal and chromatid aberrations were observed. They were 0·67 and 2%, respectively. Finally, G-banding pattern analysis of chromosomes of R. r. roxellanae were carried out. The results show that each homologous pair has its own special banding pattern, so that each of them is easily recognizable. Idiograms of chromosome complements with the Giemsa banding pattern are constructed.     

Giemsa banding pattern of the karyotype of Aotus griseimembra Elliot 1912. A preliminary study

Chromosomal polymorphism for a Robertsonian fission-fusion element is present in the karyotype of the owl monkey species Aotus griseimembra Elliot 1912. Giemsa banding reveals the homology of the chromosomes in the Robertsonian element—a large median chromosome equivalent to a large terminal and large subterminal chromosome.     

Chromosome 1 in crested and marbled newts (Triturus)

The heteromorphic chromosomes 1 of Triturus cristatus carnifex and T. marmoratus were studied in mitotic metaphase after staining with the Giemsa C-banding technique and with the fluorochromes, DAPI (AT-specific) and mithramycin (GC-specific). They were also examined in the lampbrush form under phase-contrast before fixation and after fixation and staining with Giemsa. Chromosomes 1 of T.c. carnifex are asynaptic and achiasmatic throughout most of their long arms. They are also heteromorphic in most of their long arms for the patterns of Giemsa and fluorochrome staining and the distribution of distinctive lampbrush loops. The heteromorphic regions correspond to the regions that are asynaptic and achiasmatic. They stain more strongly with mithramycin and more weakly with DAPI than the remainder of the chromosomes, signifying that their DNA is relatively rich in GC. The patterns of staining with Giemsa and fluorochromes and the distributions of distinctive lateral loops vary from one animal to another in the same species and even in the same population. The asynaptic and achiasmatic regions of chromosomes 1 in T. marmoratus extend throughout the whole of the long arms and well beyond the heterochromatic region. Chiasmata form only in the short arm and occasionally in the short euchromatic segment at the tip of the long arms. The staining patterns of chromosomes 1 in T. marmoratus differ from those in T.c. carnifex although, like carnifex, their DNA is relatively GC-rich. The chromosomes 1 of T. marmoratus are more submetacentric than those of T.c. carnifex. In T. marmoratus chromosome 1B is about 12% shorter than 1A. There is a short paracentric inversion heterozygosity in the long arm of chromosome 1B in T. marmoratus which probably accounts for the lack of chiasmata in the euchromatin that separates the centromere from the start of the heterochromatin. In both carnifex and marmoratus, embryos that are homomorphic for chromosome 1 arrest and die at the late tailbud stage of development. The same applies to F₁ hybrid embryos T.c. carnifex x T. marmoratus, and this has permitted identification of chromosomes 1A and 1B in both species. There is no correspondence between patterns of Giemsa or fluorochrome staining of the heteromorphic regions of chromosome 1 and any feature of the lampbrush chromosomes. However, the short euchromatic ends of the long arms of chromosomes 1 in both species are distinguished in the lampbrush form by a series of uniformly small loops of fine texture associated with very small chromomeres. The Giemsa C-staining patterns of both chromosomes 1A and 1B are different in each of the four subspecies of T. cristatus. T.c. karelinii stands out by having unusually large masses of Giemsa C-staining centromeric heterochromatin on all but 1 of its 12 chromosomes. A scheme is proposed for the evolution of chromosome 1 in T. cristatus and T. marmoratus, based on all available cytological and molecular data.     

The Giemsa banding patterns of the standard and four reconstructed karyotypes of Vicia faba

The Giemsa banding patterns of the standard karyotype of Vicia faba and of four new karyotypes with easily interdistinguishable chromosomes due to interchanges and inversions are described and compared with the data of other authors on preferential Giemsa staining in Vicia faba. All karyotypes contain 14 easily reproducible marker bands which characterize chromosome segments known to be heterochromatic. It is shown that the preferential Giemsa staining of chromosome regions is a valuable tool for the localization of translocation and inversion points in the chromosomes of the reconstructed Vicia karyotypes. A close correlation exists between banding patterns, segment extension by incorporation into chromosomal DNA of azacytidine and mutagen-specific clustering of induced chromatid aberrations in the new karyotypes.     

尾斑瘰螈的核型和C带研究

用常规Giemsa染色和BSG技术研究了尾斑瘰螈的核型和C带。该螈的染色体数 为2n=24,包括9对中部着丝粒染色体和3对亚中部着丝粒染色体(Nos. 7、10、11);BSG显带处理后全部染色体都显示了弱的着丝粒带,同时还显示了46条近着丝粒区插入带; 其核型和C带均不同于已研究过的国内蝾螈科动物,未发现与性别有关的异形色体。     

Activity of the endogenous RNA polymerase on fixed polytene chromosomes.

Fixed polytene chromosomes can serve as templates for RNA synthesis in situ, using the endogenous chromosomal DNA-dependent RNA polymerase. Labelling is mainly localized in band regions. However, radioactivity can also be found in interbands and puffs similar to that which occurs in vivo. It is also found by this technique that the nucleolar RNA polymerase appears to be active in these preparations and requires Mg²⁺ for activity. Since the pattern of the RNA transcribed in situ with the DNA-dependent RNA polymerase from E. coli of native chromosomes differs from that with endogenous RNA polymerase and resembles the one obtained with heat-treated chromosomes, it is suggested that the polymerase from E. coli does not act specifically on eukaryotic chromosomes.     

Dynamic aspects of trypsin-giemsa banding.

The trypsin-Giemsa banding procedure was adapted so that chromosomes could be observed through the microscope during treatment and staining. Trypsin treatment resulted only in a swelling of the chromatids. Chromosome bands which appear as raised structures with interference contrast optics emerged only after staining with Giemsa. These structures remain after Giemsa destaining, suggesting that an irreversable change in chromosome structure is induced by Giemsa. Observations of the stain flow indicate that the positioning of the chromosomes has an effect on the quality of band production. These studies also revealed that bands appear in a reproducible sequence on individual chromosomes, which suggests that alterations take place at different rates along the length of the chromosomes.     

An investigation of the mechanism of the reciprocal differential staining of BUdR-substituted and unsubstituted chromosome regions.

After treatment with hot NaH₂PO₄ at pH 9, BUdR-substituted and unsubstituted chromosome regions are palely and intensely stained with Giemsa, respectively; however, after treatment with the same solution at pH 4, the reciprocal staining patterns are produced, i.e. these chromosome regions are intensely and palely stained, respectively. The nature of the mechanisms responsible for this reciprocal differential Giemsa staining of BUdR-substituted and unsubstituted chromosome regions has been investigated by Feulgen staining, electron microscopy, and radioisotope analyses involving scintillation counting and autoradiography. The results indicate that different mechanisms are responsible for the two types of staining effect. The high pH NaH₂PO₄ treatment preferentially extracts BUdR-substituted DNA into the treatment solution, relative to unsubstituted DNA. The collective evidence from this and other work suggests that BUdR-substituted DNA in the chromosomes is partially photolysed by exposure to daylight during the harvesting procedure, and the degraded DNA is subsequently solubilized and extracted during the high pH treatment. This quantitative reduction of DNA in the BUdR-substituted chromosome regions results in pale Giemsa staining of these regions. The low pH NaH₂PO₄ treatment does not produce a significant extraction of either BUdR-substituted or unsubstituted DNA into the treatment solution; rather, there may be a redistribution of the unsubstituted DNA relative to the BUdR-substituted DNA such that the unsubstituted DNA is preferentially dispersed outside the boundaries of the chromosomes onto the surrounding area of the slide. It is suggested that the BUdR-substituted chromosome regions stain relatively intensely with Giemsa after the low pH treatment because the DNA in these regions is less dispersed than that in the unsubstituted regions.     

Identification and designation of telocentric chromosomes in barley by means of Giemsa N-banding technique

Summary Seven complete chromosomes and nine telocentric chromosomes in telotrisomics of barley (Hordeum vulgare L.) were identified and designated by an improved Giemsa N-banding technique. Karyotype analysis and Giemsa N-banding patterns of complete and telocentric chromosomes at somatic late prophase, prometaphase and metaphase have shown the following results: Chromosome 1 is a median chromosome with a long arm (Telo 1L) carrying a centromeric band, while short arm (Telo 1S) has a centromeric band and two intercalary bands. Chromosome 2 is the longest in the barley chromosome complement. Both arms show a centromeric band, an intercalary band and two faint dots on each chromatid at middle to distal regions. The banding pattern of Telo 2L (a centromeric and an intercalary band) and Telo 2S (a centromeric, two intercalary and a terminal band) corresponded to the banding pattern of the long and short arm of chromosome 2. Chromosome 3 is a submedian chromosome and its long arm is the second longest in the barley chromosome complement. Telo 3L has a centromeric (fainter than Telo 3S) and an intercalary band. It also shows a faint dot on each chromatid at distal region. Telo 3S shows a dark centromeric band only. Chromosome 4 is the most heavily banded one in barley chromosome complement. Both arms showed a dark centromeric band. Three dark intercalary bands and faint telomeric dot were observed in the long arm (4L), while two dark intercalary bands in the short arm (4S) were arranged very close to each other and appeared as a single large band in metaphase chromosomes. A faint dot was observed in each chromatid at the distal region in the 4S. Chromosome 5 is the smallest chromosome, which carries a centromeric band and an intercalary band on the long arm. Telo 5L, with a faint centromeric band and an intercalary band, is similar to the long arm. Chromosomes 6 and 7 are satellited chromosomes showing mainly centromeric bands. Telo 6S is identical to the short arm of chromosome 6 with a centromeric band. Telo 3L and Telo 4L were previously designated as Telo 3S and Telo 4S based on the genetic/linkage analysis. However, from the Giemsa banding pattern it is evident that these telocentric chromosomes are not correctly identified and the linkage map for chromosome 3 and 4 should be reversed. One out of ten triple 2S plants studied showed about 50% deficiency in the distal portion of the short arm. Telo 4L also showed a deletion of the distal euchromatic region of the long arm. This deletion (32%) may complicate genetic analysis, as genes located on the deficient segment would show a disomic ratio. It has been clearly demonstrated that the telocentric chromosomes of barley carry half of the centromere. Banding pattern polymorphism was attributed, at least partly, to the mitotic stages and differences in techniques.Contribution from the Department of Agronomy and published with the approval of the Director of the Colorado State University Experiment Station as Scientific Series Paper No. 2730. This research was supported in part by the USDA/SEA Competitive Research Grant 5901-0410-9-0334-0, USDA/ SEA-CSU Cooperative Research Grant 12-14-5001-265 and Colorado State University Hatch Project. This paper was presented partly at the Fourth International Barley Genetics Symposium, Edinburgh, Scotland, July 22–29, 1981     

The karyotype of Clivia mirabilis analyzed by differential banding and fluorescence in-situ hybridization

The karyotype of the recently described species Clivia mirabilis was analyzed by differential chromosome staining with Giemsa, chromomycin, and DAPI and by fluorescence in-situ hybridization with 5S and 45S rDNA probes. Like the other five Clivia species it was shown to have a unique karyotype, although its karyotype was similar in several respects to that of C. gardenii, differing in having only one pair of chromosomes with CMA bands compared with two pairs in C. gardenii and lacking any DAPI-positive bands. The evolutionary relationships of the species and their karyotypes are discussed.     

Chromosomal differentiation in two species of Aedes and their hybrids revealed by giemsa C-banding

Giemsa C-banding patterns in two species of mosquitoes, Aedes aegypti and Aedes mascarensis, their hybrids and backcross progeny revealed differences in the sex chromosome pair. In A. aegypti, the female determining or the m chromosome in both males and females shows a conspicuous band in the centromere region and another band in one arm. The male determining or the M chromosome is devoid of any bands. Progeny of crosses involving A. aegypti females and A. mascarensis males showed interesting albeit unexpected results. The intercalary band was suppressed in both sons and daughters. When such F₁ sons were backcrossed to A. aegypti females, a proportion of males developed into intersexes. These intersex progeny differed from the normal males in terms of their banding pattern. In the reciprocal cross (A. mascarensis female × A. aegypti male), the F₁ and the backcross progeny yielded the expected C-banding patterns. The implications of the reversible expression of the intercalary band on the A. aegypti m chromosome and its relevance to genetic regulation are discussed.     

Giemsa C-banded Karyotypes of Eight Species of Alstroemeria L. and Some of Their Hybrids

Karyotype analysis of Alstroemeria angustifolia ssp. angustifolia,A. aurea, A. inodora, A. ligtu spp. ligtu, A. magnifica ssp.magnifica, A. pelegrina, A. philippii and A. psittacina usingFeulgen-staining and Giemsa C-banding techniques revealed foreach species a characteristic chromosome morphology and C-bandingpattern. These characteristics could be used to identify manyindividual chromosomes in diploid interspecific hybrids. Besidesinterspecific variation, some degree of intraspecific variationin C-banding pattern was observed within A. angustifolia ssp.angustifolia, A. aurea, A. ligtu ssp. ligtu, A. magnifica ssp.magnifica and A. philippii . All species had large chromosomes (2 n =2 x =16) and asymmetrickaryotypes. In many species the short arms of the acrocentricchromosomes were darkly stained upon Giemsa C-banding. Thesetelomeric bands seemed satellites. B-chromosomes were observedin one species, A. angustifolia ssp. angustifolia . A variablenumber of large intercalary and telomeric C-bands was presentin the Chilean species, whereas the Brazilian species showedonly small C-bands. The differences in karyotypes suggest anearly separation of the Chilean and Brazilian species, afterwhich speciation followed different evolutionary pathways. InAlstroemeria the Giemsa C-banding technique can be valuableto plant taxonomists for unravelling species relationships. Alstroemeria ; Inca lily; evolution; Giemsa C-banding; karyotype     

Chromosomes of the house-cricket,Acheta domestica (L.) (Orthoptera) — Delhi population

Chromosomes of Acheta domestica were studied employing conventional Giemsa staining and fluorescence banding methods. The Delhi population seems to be characteristic with regard to the distribution of the C-bands and centromeric positions in the chromosomes and differs from those of European and American populations. Further, on the basis of base-specific fluorescence studies, it is speculated that the A-T/G bases are probably evenly distributed in the chromosomes.     

The chromosomes of CHO,an aneuploid Chinese hamster cell line: G-band,C-band,and autoradiographic analyses

Chinese hamster ovary cells (line CHO) have been used extensively for metabolic, genetic, and radiobiological studies with only a superficial appreciation for the degree of aneuploidy characteristic of the line. A thorough karyologic analysis of CHO chromosomes using autoradiographic replication patterns, as well as centromere band (C-band) and Giemsa band (G-band) analysis, is presented. Our results demonstrate that only 8 of the 21 CHO chromosomes are normal when compared with euploid Chinese hamster chromosomes. In the 13 altered chromosomes, we found evidence of translocations, deletions, and pericentric inversions. These altered chromosomes have been characterized with respect to both origin and destination of translocated material. With the exception of the X₂ chromosome, essentially all of the euploid chromatin is present in CHO cells. Autoradiographic replication patterns show that the normal sequence of chromosomal DNA synthesis is altered. Some sites which replicate late in euploid cells replicate early in CHO, and several late-replicating chromosomes in CHO cells replicate in early- or mid-S in euploid material. These studies may serve to elucidate the observed differences in mutagenic behavior between euploid fibroblasts and CHO cells.