Cell synchronization and dynamic G-banding of equine chromosomes by bromodeoxyuridine

Both dynamic G-banding and cell synchronization produced by bromodeoxyuridine (BrdU), were applied to equine chromosomes. BrdU incorporated during the first half of the S-phase is taken up into the R-bands that are early replicating. These bands, which have incorporated BrdU, cannot contract as usual and remain elongated; only the other regions of the chromosome, i.e., the G-bands, contract normally and are sharply defined. BrdU also can be used for cell synchronization. The addition of BrdU in a high concentration, 15 hours before harvest, and its removal 11 hours later, has two effects: initially the BrdU is incorporated during the first part of the S-phase and then it blocks the cells at mid-S-phase. Within the cell cycle, mid-S-phase appears to be the most vulnerable time to various blocking agents. To differentiate the regions of BrdU incorporation from those that have not been substituted, the fluorescence-photolysis-Giemsa (FPG) technique was applied as modified for horse chromosomes. This dynamic technique, which produces many prometaphase and prophase chromosomes showing very sharp G-bands, is certain to enhance the accuracy of cytogenetic analysis and aid in the standardization of equine chromosomes.     

Idiograms of horse chromosomes at prometaphase, early metaphase, and midmetaphase after R-banding by BrdU incorporation followed by the fluorochrome-photolysis-Giemsa technique

We present three idiograms of equine chromosomes, R-banded after BrdU incorporation and stained by the fluorochrome-photolysis-Giemsa technique. The haploid set of prometaphasic chromosomes shows 591 bands (range 7-38 per chromosome), the early metaphasic set 404 (range 5-26), and the midmetaphasic set 272 (range 3-18). Following cell synchronization with thymidine, more than twice as many R-bands were revealed on the resulting prometaphasic chromosomes, making possible the establishment of a very accurate and characteristic representation of this banding pattern in the domestic horse.     

The human ubiquitin-activating enzyme E1 gene (UBE1) mapped to band Xp11.3----p11.23 by fluorescence in situ hybridization.

The human gene for ubiquitin-activating enzyme E1 (UBE1) was localized by a direct mapping system that combined fluorescence in situ hybridization with replicated R-bands on prometaphase chromosomes. The fluorescent signals were localized to Xp11.3----p11.23. Simple procedures for the detection of R-bands are also described.     

FISH mapping of the IGF2 gene in horse and donkey—detection of homoeology with HSA11

Three genomic subclones derived from a phage clone containing the equine IGF2 gene were used to FISH map the gene on horse (ECA) and donkey (EAS) metaphase chromosomes. The gene mapped on ECA 12q13 band and is the first locus mapped to this horse chromosome. In donkey the gene mapped very terminal on the long arm of one small submetacentric chromosome that shows almost identical DAPI-banding pattern with ECA12. This is the first locus mapped in donkey genome. Cross species chromosome painting of equine metaphase chromosomes with human Chromosome (Chr) 11-specific probe showed homoeology of this human chromosome with ECA12 and ECA7. The novel ECA12 comparative painting results are thus in accordance with the localization of the equine IGF2 gene. Comparison of the hitherto known physical locations of IGF2 in different species, viz. human, cattle, sheep, horse, and donkey, shows that this gene tends to maintain a terminal location on the chromosome arm. Received: 12 January 1997 / Accepted: 17 March 1997     

The centromere index and relative length of human high-resolution G-banded chromosomes

Summary The relative length and centromere index were compared in prometaphase and midmetaphase for each human chromosome from five normal men. There were very few differences between prometaphase and midmetaphase chromosomes in these two parameters. Chromosome 7 had a significantly different centromere index between prometaphase and midmetaphase, but no difference in relative length. This was accounted for by significant differences in the relative length of both 7p and 7q between prometaphase and midmetaphase; 7p became relatively less condensed and 7q relatively more condensed with progression from prometaphase to midmetaphase. For chromosome 1, the short arm was significantly longer than the long arm in both prometaphase and midmetaphase, a finding that underscores the structural similarity of this chromosome among the hominids.     

Giemsa staining of the sites replicating DNA early in human lymphocyte chromosomes.

A timetable for the initiation of DNA replication in human lymphocyte chromosomes has been established by a technique which allows detection of areas of chromosomes replicating at a given interval of the S-phase. The resolution of the method, using 33258 Hoechst-Giemsa staining, is more refined than that obtained with 3H-thymidine autoradiography. Early replicating regions coincide with R-bands. The timetable is rather coarse since replication may start asynchronously in the same region of homologous autosomes of the same metaphase and since even the sequence of bands appearing on individual chromosomes sometimes deviates from the rule.     

Studies on early replication patterns in the marsupial Sminthopsis crassicaudata: no evidence for XY replication homologies

We present here the first detailed replication banding study of a marsupial species using the BrdU-replication technique. A comparison of the structural and replication bands of the chromosomes of Sminthopsis crassicaudata clearly demonstrates that the replication behavior is the same as the described for the chromosomes of eutherians. The early replicating segments correspond to R-bands, whereas the late-replicating regions tend to be situated within Q- and C-bands. Use of this technique clearly reveals an early and late replicating X chromosome. The very small Y chromosome can be subdivided into two replication segments, but no replication homologies can be demonstrated between the X and Y chromosomes of S. crassicaudata.     

江苏櫓稻核型的初步分析

本文研究了江苏穭稻——一种草型栽培稻(Orysa sativa L.)的核型与染色体带型。结果表明,穭稻的核型有6对中位染色体(K_1,K_3,K_6,K,K(?)和K_(11)),4对亚中位染色体(K_2,K(?)K_0和K(?)),1对亚端位染色体(K_4)和1对随体染色体(K_(10)),在K_3染色体的长臂上有一次缢痕。Giemsa分带处理表明,穭稻染色体带型比较丰富,各条染色体的带型特征明显。本文还讨论了江苏穭稻与普通野生稻和栽培稻在核型及染色体带型上的异同。     

R-banding and nonisotopic in situ hybridization: precise localization of the human type II collagen gene (COL2A1)

Summary A new mapping system, based on nonisotopic in situ hybridization combined with fluorescent staining of replicated prometaphase R-bands, is described. Replication of the bands is achieved by treatment of thymidinesynchronized cells with bromodeoxyuridine. The human COL2A1 gene was mapped to band 12q13.11–q13.12 in this manner, to illustrate the potential of the technique for improving the precision of chromosomal mapping and physical ordering of genes.     

Lemur chromosomal study with simultaneous R-banding and chromosome painting

A method for simultaneously obtaining R-banding and chromosome painting is described. It combines fluorescence in situ hybridization with replication of R-bands by 5-bromo-2′-deoxyuridine incorporation into synchronized cells. Distinctive R-banding induced by a modified fluorochrome-photolysis procedure can be observed on both painted and non-painted chromosomes. This method applied to Lemur chromosomes was developed for further studies of chromosomal changes in the evolution of prosimian primates and could also be used in other cytogenetic applications where simultaneous identification of chromosomal R-bands and hybridization signal is needed. Received: 14 September 1999; in revised form: 18 October 1999 / Accepted: 20 October 1999     

Chromosome mapping of the human cytidine-5′-triphosphate synthetase (CTPS) gene to band 1p34.1–p34.3 by fluorescence in situ hybridization

Summary The human cytidine-5-triphosphate synthetase (CTPS) gene was mapped by a direct mapping system combined with fluorescence in situ hybridization and replicated prometaphase R-bands. By high-resolution banding analysis, the signals were localized to band 34.1–34.3 of the short arm of chromosome 1; 1p34.1–p34.3. Simple procedures for the detection of R-bands are described.     

An immunoperoxidase technique for demonstrating antilymphocytic globulin on cell surfaces

Summary An immunoperoxidase technique is described, for detecting horse IgG on human lymphocytes incubated with equine antilymphocytic globulin (ALG). Using this method, horse IgG was demonstrated on lymphocytes which had been incubated with ALG in dilutions of up to approximately 1:1,500.     

Comparative mapping of 18 equine type I genes assigned by somatic cell hybrid analysis

Polymerase chain reaction primers designed from horse cDNA sequences and from consensus sequences highly conserved in mammalian species were used to amplify markers for synteny mapping 18 equine type I genes. These markers were used to screen a horse–mouse somatic cell hybrid panel (UCDavis SCH). Fourteen primer sets amplified horse-specific fragments, while restriction enzyme digests of PCR products were used to distinguish the fragments amplified from horse and mouse with four primer sets. Synteny assignments were made based on correlation values between each marker tested and other markers in the UCDavis SCH panel database. The 18 horse genes were assigned to previously established synteny groups. Synteny mapping of two genes previously mapped in the horse by FISH was used to anchor two UCD synteny groups to horse chromosomes. Previous chromosome assignments of three equine loci by FISH were confirmed. Comparative mapping analysis based on published human–horse Zoo-FISH data and the synteny mapping of 14 horse genes confirmed the physical assignment of 12 synteny groups to the respective horse chromosomes and was used to infer the physical location of one synteny group. Received: 24 July 1998 / Accepted: 29 October 1998     

Harlequin banding and localisation of sister-chromatid exchanges.

Harlequin banding (HB) was standardised on Indian muntjac chromosomes by superimposing harlequin staining or sister-chromatid differentiation and G-banding after incorporation of bromodeoxyuridine (BrdU) or cholorodeoxyuridine (CldU), and after treatment with BrdU plus mitomycin C (MMC). SCEs were localized on these chromosomes with the aid of the G-band map. There were more SCEs in G-bands than in R-bands in BrdU-incorporated chromosomes. CldU-incorporated chromosomes, however, did not show a preferential localization of SCEs in either G- or R-bands. When BrdU + MMC-induced SCEs were localized in harlequin-banded chromosomes, there was a significantly greater number of SCEs in R-bands; and there was a concomitant reduction in the frequency of SCEs in G-bands, as compared to the SCEs observed in this region after BrdU incorporation alone. Centromeric regions of chromosomes 1 and X had preferred sites for occurrence of SCEs in BrdU-incorporated chromosomes, the preferred sites being more in G-bands after BrdU and CldU incorporation and in R-bands after treatment of BrdU-incorporated chromosomes with MMC. Thus the formation of SCEs is not restricted by structure per se as defined by euchromatin or heterochromatin, but depends on the site of lesion production, type of lesion and repair pathway followed.     

Synchronization of human lymphocyte cultures by fluorodeoxyuridine

A simple technique for synchronization of human lymphocyte cultures with fluorodeoxyuridine (FudR) is presented. The S-phase block induced by the FudR is released by simultaneous exposure to 5-bromodeoxyuridine (BrdU) and Hoechst 33258 or by thymidine and Hoechst 33258. This method provides a high mitotic index with high percentage of prometaphase chromosomes. This simple method is highly advantageous and easy to utilize in clinical cytogenetics.     

In situ random primer extension of metaphase chromosomes

We have developed a technique of random primer extension of fixed chromosomes that is applicable to both mouse and man. Human chromosomes are not homogeneously labeled with this technique; those regions corresponding to R-bands appear to be more sensitive than those identified as G-bands, whereas centromeric regions are not labeled. These results not only corroborate specific structural differences between distinct regions of mammalian genomes but also open up the possibility of assays with specific primers to test whether primer extension is useful for the identification of genes and families of sequences on chromosomes.     

Differences in the structural organization of the G- and R-bands detectable during the differential decondensation of chromosomes

The ultrastructure of G- and R-bands in differentially decondensed chromosomes of Chinese hamster was studied with a gradual decrease in CaCl2 concentration in the medium. The gradual reduction of CaCl2 concentration leads to the decondensation of compact G-bands into chromonemes, chromomeres and further into DNP-fibrils. In the complete local decondensation zones (R-bands), the DNP-fibril orientation is parallel to the chromosome longitudinal axis. These zones have no lateral loops or chromomeres. Thus, different chromosome regions corresponding to G- and R-bands possess different sensibility to the decondensing action. Following the complete decondensation in the calcium-free medium chromosomes can be "reconstructed" by adding Ca2+. The data obtained permit to suggest a "fastener" model of the mitotic chromosome organization in which the chromosome represents an hierarchy of discrete structures--G-bands, chromomeres, nucleomeres (superbeads) and nucleosomes. The structural integrity of these levels is supported by specific protein "fasteners".     

Chromatin configuration and epigenetic landscape at the sex chromosome bivalent during equine spermatogenesis

Pairing of the sex chromosomes during mammalian meiosis is characterized by the formation of a unique heterochromatin structure at the XY body. The mechanisms underlying the formation of this nuclear domain are reportedly highly conserved from marsupials to mammals. In this study, we demonstrate that in contrast to all eutherian species studied to date, partial synapsis of the heterologous sex chromosomes during pachytene stage in the horse is not associated with the formation of a typical macrochromatin domain at the XY body. While phosphorylated histone H2AX (γH2AX) and macroH2A1.2 are present as a diffuse signal over the entire macrochromatin domain in mouse pachytene spermatocytes, γH2AX, macroH2A1.2, and the cohesin subunit SMC3 are preferentially enriched at meiotic sex chromosome cores in equine spermatocytes. Moreover, although several histone modifications associated with this nuclear domain in the mouse such as H3K4me2 and ubH2A are conspicuously absent in the equine XY body, prominent RNA polymerase II foci persist at the sex chromosomes. Thus, the localization of key marker proteins and histone modifications associated with the XY body in the horse differs significantly from all other mammalian systems described. These results demonstrate that the epigenetic landscape and heterochromatinization of the equine XY body might be regulated by alternative mechanisms and that some features of XY body formation may be evolutionary divergent in the domestic horse. We propose equine spermatogenesis as a unique model system for the study of the regulatory networks leading to the epigenetic control of gene expression during XY body formation.     

A structural basis for R- and T-banding: a scanning electron microscopy study

The structure of reverse (R)-banded and telomeric (T)-banded chromosomes was studied by examination of the same chromosomes first in the light microscope (LM) followed by the scanning electron microscope (SEM). This procedure demonstrated a structural basis to both the R- and T-banding techniques. A direct correlation was shown between the LM staining patterns and the structural patterns observed in the SEM. In the R-banded chromosomes the positively stained R-bands, viewed by LM, corresponded to highly fibrous three-dimensional regions in the SEM. The negatively stained R-interbands corresponded to flatter regions from which material appeared to have been extracted. These structural observations strongly support the suggestion that chromosomal material is preferentially lost from the R-interbands with aggregation of fibres in the R-bands. T-banded chromosomes showed a similar structure to the R-banded chromosomes. The positively stained T-bands located at the telomeres corresponded to regions of highly aggregated fibres. The remainder of the chromosome, corresponding to the negatively stained area, had a flattened and extracted appearance. These similarities in morphology between the T- and R-banded chromosomes support the view that T-bands result from a progressive breakdown of the R-banded chromosome structure.