Chromosome banding in Amphibia. XXVII. DNA replication banding patterns in three anuran species with greatly differing genome sizes

The mitotic chromosomes of three anuran species, Scaphiopus holbrooki, Litoria infrafrenata and Odontophrynus americanus, were analyzed by means of the 5-bromodeoxyuridine/deoxythymidine (BrdU/dT) replication banding technique. These species exhibit large differences in their genome sizes: S. holbrooki possesses one of the smallest genomes among vertebrates, L. infrafrenata has a genome size near the modal DNA value of most Amphibia, whereas O. americanus is a tetraploid species. BrdU/dT labeling induces reproducible and reliable R- and G-replication bands along the metaphase chromosomes of all three species. Irrespective of the genome size of the species considered, the number of early (R-) and late (G-) replicating bands per haploid karyotype is nearly the same. The chromosomes of the autotetraploid O. americanus can be arranged into sets of four homologous chromosomes (quartets). C-bands and BrdU/dT replication bands reveal heterogeneity within the quartets 1, 3 and 4 that are interpreted as the initiation of a diploidization process.     

斜鳞蛇核型高分辨显带及减数分裂的观察

本文报道了斜鳞蛇的核型和银带。二倍体数目２ｎ－３６,其中包括８对８对大染色体（６对中着丝粒染色体,２对亚中着丝粒染色体）和１０对小梁色体。其核仁组织区（ＮＯＲｓ）位于第１对小梁色体末端。并采用ＴｄＲ－ＢｒｄＵ处理方法,显示斜鳞蛇复制带,实验证明,用大剂量的胸腺嘧啶核苷阻断细胞周期,再以ＢｒｄＵ渗入Ｓ期细胞中复制的染色体区域,并显示带纹。同时还斜鳞蛇的减数分裂进行了观察。     

纳特竖蟾(无尾目:滑背蟾科) 的细胞遗传、与相关属物种的核型相似性并兼论其分类地位

本文报道纳特竖蟾(Eupemphix nattereri Steindachner,1863)巴西3个产地标本的核型,均为2n=22,由1对端部和10对中或亚中部着丝点染色体组成.仅一对NORs位于No.11染色体对长臂端部,而其位置有别于常见的具有标志性意义的近着丝点位置.该NORs的位置为rDNA为探针的荧光原位杂交(FISH)所确认.各染色体对中,着丝点C-带明显,插入或端带偶见.某些标本的No.8同源染色体对间C-带的大小随异染色质化的程度不同而变化.居于较小的实验标本量,这种在3个产地的雌或雄性标本之一中观察到的C-带异型现象可能为种下细胞地理学变异,亦或为细胞学意义的性染体分化.3产地之一的标本核型的No.11着丝点C-带异染色质化的程度较高.CMA3染色检测到部分GC-rich区域,DAPI染色未显示任何AT-rich区.成功获得BrdU复制带,并将其与滑背蟾类动物(leiuperid)中近缘属及物种进行对比分析.比较结果表明,纳特竖蟾的核型与斑符泡蟾种组(Physalaemus signifer group)难以相互区别,而与肿肋蟾属(Pleurodema Tschudi,1838)极为相似.核型数据支持形态学上将纳特竖蟾、二光肿肋蟾[Pleurodema diplolister (Peters,1870)]和短头肿肋蟾[P.brachyops(Cope,1869)]划为同一分支的观点[动物学报 53(2):285-293,2007].     

Replication timing properties across the pseudoautosomal region boundary and cytogenetic band boundaries on human distal Xp

The establishment of human chromosomal regions as distinct and characteristic domains has been demonstrated by the reproducible banding patterns observed on metaphase chromosomes as a result of various staining techniques. Although the exact molecular properties responsible for the patterns are not well understood, a general correlation has been established between the time of replication of a particular region of DNA and its banding characteristics. Using a replication timing assay based on fluorescence in situ hybridization patterns, we investigated replication timing properties across chromosomal regions with potentially distinct chromatin properties. Relative replication timing values were determined using cosmid DNA probes around the pseudoautosomal region boundary in Xp22.3 and the cytogenetic band boundary regions surrounding Xp22.2. Although we observed replication timing domains that were generally consistent with cytogenetic banding patterns, we did not find sharp replication timing boundaries at either the pseudoautosomal region boundary or at the cytogenetic band boundaries. Received: 6 September 1997; in revised form: 16 December 1997 / Accepted: 5 January 1998     

Cytogenetics of three breeds of river buffalo (Bubalus bubalis L.), with evidence of a fragile site on the X chromosome

The cytogenetic study of 182 river buffalo (Bubalus bubalis L., 2n = 50) of Murrah, Mediterranean and Jaffarabadi breeds, from the State of S?o Paulo, was carried out to characterize their chromosomes and to detect possible chromosomal abnormalities. The karyotypes were indistinguishable with conventional staining as well as with C and replication R banding techniques. In about 44% of the sample (8 males and 72 females), an X marker chromosome due to a fragile site was shown. The frequency of metaphases expressing the fragility site on the X was highly variable, from 2.86 to 41.03%. In females, the fragile site, rarely appeared on both X chromosomes. Most of the metaphases showed only 1 marker chromosome. In R-banded metaphases using 5-bromodeoxyuridine (BrdU) treatment, it corresponded in general to the late replicating X chromosome. No correlation between the X fragile site and altered phenotype was found. Structural and numerical chromosome rearrangements were ruled out in the present sample of buffalo.     

家蚕染色体复制区带的初步显示

该文采用家蚕Bomoyx mori活体注射BrdU结合FPG(fluorochrome photolyusis Giem-sa)显带方法,以生殖腺为材料,成功显示出家蚕有丝分裂中期染色体复制带。由于处于S-期的细胞有早有晚,且同一细胞DNA各片段的复制亦有先后,因此BrdU掺入DNA合成的时间也有所不同,从而可产生出早、中、晚复制带型。BrdU掺入时间早,则会在家蚕部分染色体上出现大面积浅染带纹的早复制带。每一染色体皆有其独特的带纹特征,据此可初步将它与其它染色体相互区分;随着BrdU掺入时间的推后,染色体上会出现深浅交替、丰富的带纹,即中复制带型;至S-期DNA合成晚期掺入BrdU,最终染色体出现以深染带纹为主,浅染带纹仅出现于少数染色体的中部、近中部或端部的晚复制带。     

Detection of bromodeoxyuridine-incorporation in mammalian chromosomes by a bromodeoxyuridine-antibody

Summary We have shown that a commercially available antibody allows the demonstration of bromodeoxyuridine (BrdU) incorporation into mammalian chromosomes by the typical immunologic staining techniques. the replication patterns obtained are comparable to those after BrdU-incorporation and FPG-staining, though with reversed staining specificity. The quality of the patterns equals that of commonly used differential staining but the sensitivity of BrdU-detection is superior. Thus the new technique may be very promising for all cytogenetic studies concerning any kind of chromosome replication.     

大熊猫与黑熊显带染色体的比较研究

以体外培养的大熊猫（Ａｉｌｕｒｏｐｏｄａｍｅｌａｎｏｌｅｕｃａ）与黑熊（Ｓｅｌｅｎａｒｃｔｏｓｔｈｉｂｅｔａｎｕｓ）外周血淋巴细胞为实验材料,应用ＢｒｄＵ复制带显示技术,研究了大熊猫和黑熊染色体晚复制带带型。通过对大熊猫与黑熊显带染色体带型的比较,发现黑熊部分具端着丝粒的染色体与大熊猫部分具中,亚中,或亚端着丝粒的染色体的整个短臂或整个长臂有明显的带型相似性,在黑熊具中,亚中着丝粒染色体中,仅３３     

Chromosome banding patterns of four species of bats, with special reference to a case of X-autosome translocation

The karyotypes of 4 species of bats, Artibeus lituratus (Phyllostomatidae), Pipistrellus pipistrellus (Vespertilionidae), Pteropus alecto and P. giganteus (Pteropodidae), were studied after several banding techniques. For A. lituratus, in which an X-autosome translocation was observed, an analyse of the replication pattern in the rearranged chromosome was also made after BrdU incorporation.     

Karyotype differentiation in Chromaphyosemion killifishes (Cyprinodontiformes, Nothobranchiidae). III: extensive karyotypic variability associated with low mitochondrial haplotype differentiation in C. bivittatum

We investigated chromosomal evolution in the African killifish species Chromaphyosemion bivittatum using a combination of cytogenetic and phylogenetic methods. Specimens from five populations were examined by conventional Giemsa staining as well as sequential chromosome banding with 4',6-diamidino-2-phenylindole (DAPI), chromomycin A(3) (CMA(3)), AgNO(3)-staining and C-banding. The cytogenetic analysis revealed variability in 2n ranging from 2n = 29 to 2n = 36 and in NF ranging from NF = 38 to NF = 44. Two populations showed an extensive chromosomal polymorphism (2n = 29-34, NF = 44 and 2n = 32-34, NF = 38-42, respectively). Karyotypic variability within and among populations was mainly due to Robertsonian translocations and heterochromatin additions, and chromosome banding patterns suggested that both types of chromosomal rearrangements were related to the presence of AT-rich heterochromatin. A phylogenetic analysis of the partial mitochondrial (mt) cytochrome b gene, using specimens from eleven populations, revealed a low degree of haplotype differentiation, which suggested a relatively recent divergence of the populations examined. This finding conformed to the low degree of morphological differentiation observed among C. bivittatum populations and might indicate fast chromosomal evolution. The high karyotypic variability may be caused by an elevated chromosomal mutation rate as well as certain aspects of the mating system and population dynamics of C. bivittatum facilitating the fixation of new chromosomal variants.     

Replication banding patterns in the spined loach,Cobitis taenia L. (Pisces,Cobitidae)

The chromosomal complement of Cobitis taenia was analysed by replication banding techniques to determine whether there were specific patterns that could allow distinction of the different chromosomes. The diploid chromosome number of 2n = 48 is diagnostic of this species. In vivo 5-bromodeoxyuridine (5-BrdU) incorporation induced highly reproducible replication bands. Most of the chromosome pairs were distinguishable on the base of their banding patterns. The karyotype, consisting of five pairs of metacentrics, nine pairs of submetacentrics and 10 pairs of subtelocentrics and acrocentrics, was confirmed. C-banding and replication banding patterns were compared, and heterochromatin was both early and later replicating. C-positive heterochromatin in centromeric regions was mainly early replicating, but that located in pericentromeric regions was late replicating. Most of the late-replicating regions found interstitially were C-band negative. The results obtained so far for combined chromosomal staining methods of C. taenia and other Cobitis fish species are discussed.     

Analysis of chromosome replication by a BrdU antibody technique

Chromosome replication was studied without synchronization in human lymphocyte and amniotic cell cultures visualizing very short 5-bromodeoxyuridine (BrdU) pulses by an immunologic technique (BAT). The findings agree in general with those facts known from earlier BrdU staining techniques. The very high sensitivity of BAT was shown to allow the detection of replication in a band where 1 in 200 nucleotides is replaced by BrdU. The main observations are: though the replication patterns after BAT appear strange the bands correspond to those described by the Paris Conference (1971). At the beginning of the S-phase a stepwise onset of replication in only a subset of R-bands is confirmed. There is a considerable difference in the sensitivity between early and late S (S_E and S_L) for the detection of BrdU pulses. This difference probably reflects a different spatial arrangement of chromatin in R-bands as compared with G-bands below the level of cytogenetic analysis. The use of short pulses did not reveal any additional subdivision of S_E or S_L. The correspondence between chromosomal bands and replicon clusters is discussed briefly with respect to the different time they need for replication.     

Replication Banding in two Mediterranean Moray eels: Chromosomal Characterization and Comparison

Early and late replication bandings have been obtained by in vitro BrdU incorporation in the Mediterranean Muraenidae species Muraena helena and Gymnothorax unicolor, and used to characterize their karyotypes. A comparative analysis of the banding patterns allowed to point out high karyotype similarity as well as chromosome rearrangements that occurred in karyotype evolution between these species.     

On the mechanism of differential Giemsa staining of bromodeoxyuridine-substituted chromosomes

Summary Experiments were performed to find out whether different mechanisms are involved in FPG-(fluorescent plus Giemsa) staining for the demonstration of replication patterns and sister chromatid differentiation (SCD) after bromodeoxyuridine (BrdU)-substitution of V79 Chinese hamster chromosomes. The influence of variations of the staining procedure on the quality of both SCD and replication patterns was comparatively investigated and differences in the demonstration of these two phenomena within the same chromosome were studied using various BrdU-labeling protocols. The results show that at least graduated differences exist. For a good differentiation of replication patterns a stronger FPG-treatment is necessary than it is for SCD. Partial BrdU substitution only leads to replication patterns in the next mitosis. A further round of replication either in the presence or absence of BrdU causes a reduced staining of the complete chromatid and three-way differentiation is seen in third generation mitoses. These results support the view that alterations of chromosomal proteins during BrdU-incorporation and replication of BrdU-substituted DNA are decisive for differential staining.     

Comparative karyotype analysis and chromosome evolution in the genus Aplastodiscus (Cophomantini, Hylinae, Hylidae)

ABSTRACT: BACKGROUND: The frogs of the Tribe Cophomantini present, in general, 2n?=?24 karyotype, but data on Aplastodiscus showed variation in diploid number from 2n?=?24 to 2n?=?18. Five species were karyotyped, one of them for the first time, using conventional and molecular cytogenetic techniques, with the aim to perform a comprehensive comparative analysis towards the understanding of chromosome evolution in light of the phylogeny. RESULTS: Aplastodiscus perviridis showed 2n?=?24, A. arildae and A. eugenioi, 2n?=?22, A. callipygius, 2n?=?20, and A. leucopygius, 2n?=?18. In the metaphase I cells of two species only bivalents occurred, whereas in A. arildae, A. callipygius, and A. leucopygius one tetravalent was also observed besides the bivalents. BrdU incorporation produced replication bands especially in the largest chromosomes, and a relatively good banding correspondence was noticed among some of them. Silver impregnation and FISH with an rDNA probe identified a single NOR pair: the 11 in A. perviridis and A. arildae; the 6 in A. eugenioi; and the 9 in A. callipygius and A. leucopygius. C-banding showed a predominantly centromeric distribution of the heterochromatin, and in one of the species distinct molecular composition was revealed by CMA3. The telomeric probe hybridised all chromosome ends and additionally disclosed the presence of telomere-like sequences in centromeric regions of three species. CONCLUSIONS: Based on the hypothesis of 2n?=?24 ancestral karyotype for Aplastodiscus, and considering the karyotype differences and similarities, two evolutionary pathways through fusion events were suggested. One of them corresponded to the reduction of 2n?=?24 to 22, and the other, the reduction of 2n?=?24 to 20, and subsequently to 18. Regarding the NOR, two conditions were recognised: plesiomorphy, represented by the homeologous small-sized NOR-bearing pairs, and derivation, represented by the NOR in a medium-sized pair. In spite of the apparent uniformity of C-banding patterns, heterogeneity in the molecular composition of some repetitive regions was revealed by CMA3 staining and by interstitial telomeric labelling. The meiotic tetravalent might be due to minute reciprocal translocations or to non-chiasmatic ectopic pairing between terminal repetitive sequences. The comparative cytogenetic analysis allowed to outline the chromosome evolution and contributed to enlighten the relationships within the genus Aplastodiscus.     

Molecular and classical cytogenetic analyses demonstrate an apomorphic reciprocal chromosomal translocation in Gorilla gorilla.

The existence of an apomorphic reciprocal chromosomal translocation in the gorilla lineage has been asserted or denied by various cytogeneticists. We employed a new molecular cytogenetic strategy (chromosomal in situ suppression hybridization) combined with high-resolution banding, replication sequence analysis, and fluorochrome staining to demonstrate that a reciprocal translocation between ancestral chromosomes homologous to human chromosome 5 and 17 has indeed occurred.     

The Unique Karyotype of Henochilus wheatlandii, a Critically Endangered Fish Living in a Fast-Developing Region in Minas Gerais State, Brazil

Henochilus wheatlandii, the only species of this genus, is critically endangered and was considered extinct for over a century. The rediscovery of this fish in 1996 made it possible to study its phylogenetic relationships with other species in the subfamily Bryconinae. The aim of this study was to characterise the karyotype of H. wheatlandii. Standard staining, C-positive heterochromatin and nucleolar organiser region (NOR) banding, chromomycin A(3) staining, and fluorescent in situ hybridisation (FISH) using 5S rDNA and 18S rDNA probes were conducted on nineteen specimens collected in the Santo Antonio River, a sub-basin of the Doce River in Ferros municipality, Minas Gerais State, Brazil. Henochilus wheatlandii shared the same diploid number and chromosome morphology as other species of Bryconinae. However, its heterochromatin distribution patterns, NOR localisation, and FISH patterns revealed a cytogenetic profile unique among Neotropical Bryconinae, emphasizing the evolutionary uniqueness of this threatened species.     

Differential giemsa staining of sister chromatids after extraction with acids

Chromosomes of Chinese hamster strain cells were air-dried on slides after BrdU substitution for two or three rounds of replication. The preparations were treated with 20% PCA at 55 degrees C for 20-30 min, or 5N HCl at 55 degrees C for 15-20 min. After staining with Giemsa, unifilarly BrdU-substituted chromatids stained faintly and bifilarly substituted chromatids stained darkly. Such a pattern of sister chromatid differential staining was confirmed by the examination of metaphase cells grown with BrdU for three rounds of replication.     

Chromosome banding and DNA replication patterns in bird karyotypes

The karyotypes of the domestic chicken (Gallus domesticus), Japanese quail (Coturnix coturnix), and griffon vulture (Gyps fulvus) were studied with a variety of banding techniques. The DNA replication patterns of bird chromosomes, analyzed by incorporation of 5-bromodeoxyuridine (BrdU) and deoxythymidine (dT), are presented here for the first time. In particular, the time sequence of replication of the ZZ/ZW sex chromosomes throughout the S-phase was meticulously analyzed. BrdU and dT incorporation are very useful methods to identify homoeologies between karyotypes, as well as rearrangements that occurred in the macroautosomes during speciation. The Z chromosomes of the three birds displayed the same replication patterns, indicating a high degree of evolutionary conservation. In the homogametic male, BrdU and dT incorporation revealed no evidence of asynchronous replication between euchromatic bands in the ZZ pair. The same was true of the three Z chromosomes in a triploid-diploid chimeric chicken embryo. Minor replication asynchronies between the homologous ZZ or ZZZ chromosomes were restricted to heterochromatic C-bands. These results confirm that, in the ZZ male/ZW female sex-determining system of birds, dosage compensation for Z-linked genes does not occur by inactivation of one of the two Z chromosomes in the homogametic male. The heterochromatic W chromosomes of the three species showed bright labeling with distamycin A/mithramycin counterstain-enhanced fluorescence and exhibited significantly delayed DNA replication. The nucleolus organizers of birds, frequently located in microchromosomes, were also distinguished by bright distamycin A/mithramycin fluorescence.