限制酶AluI显带和CA_3/DA/DAPI染色表达的家猪染色体结构异染色质

采用限制酶AluI显带、CA_(?)/DA/DAPI荧光染色和常规C带技术研究了家猪染色体着丝粒结构异染色质,结果表明:着丝粒结构异染色质至少可被区分为3类,并且在染色体组内各有其特异的染色体分布。将家猪染色体DA/DAPI荧光带和限制酶AluI显带与人类染色体比较,发现家猪13—18号端着丝粒染色体显带特征与人染色体1,9、16、Y一致。提示家猪13—18号端着丝粒区结构异染色质存在与人类随体DNA相似的DNA组成。     

Fifty years of cytogenetics: a parallel view of the evolution of cytogenetics and genotoxicology

A parallelism exists between human cytogenetics and cytogenetic toxicology. The breakthroughs, mostly coming from and used in clinical genetics, are widely used in genetic toxicology. The birth of human cytogenetics occurred in 1956 when it was published that the diploid number of chromosomes in humans is 46. The first stage in chromosome-induced mutagenesis began in 1938 when Sax published the effects of X-rays on the chromosomes of Drosophila. In 1959, the cytogenetic anomalies for Down, Klinefelter, and Turner syndromes were described, and parallelly in 1960, the first publication on chromosomal aberrations in man caused by ionizing radiation appeared. The cytogenetic analysis of chromosomal aberrations in cell cultures is considered one of the primary methods to evaluate induced mutagenesis. At the end of the 1960s, banding techniques allowed chromosomes to be individually identified, in parallel, the sister chromatid exchange analysis technology was described. Another milestone in the history of induced mutagenesis was the discovery that mutagenic agents were able to alter chromosomal division and segregation in gonads inducing meiotic nondisjunction. Here we review new approaches and applications such as biological dosimetry, translocation scoring using FISH, and micronucleus test. Chromosomal aberrations and micronucleus test are now effective cytogenetic biomarkers of early effect used as cancer predictors. Human cytogenetics has proven to be effective over its 50-year lifespan and, although each new technique that has appeared seemed to announce its end, the fact is that the current state of cytogenetics is in reality a collection of techniques that, while common, are cheap, fast, and wide-ranging. Therefore, in genotoxicology, they continue to be useful to identify mutagenic agents as well as to evaluate and analyze exposed populations.     

Flow cytogenetics and chromosome sorting

This review of flow cytogenetics and chromosome sorting provides an overview of general information in the field and describes recent developments in more detail. From the early developments of chromosome analysis involving single parameter or one color analysis to the latest developments in slit scanning of single chromosomes in a flow stream, the field has progressed rapidly and most importantly has served as an important enabling technology for the human genome project. Technological innovations that advanced flow cytogenetics are described and referenced. Applications in basic cell biology, molecular biology, and clinical investigations are presented. The necessary characteristics for large number chromosome sorting are highlighted. References to recent review articles are provided as a starting point for locating individual references that provide more detail. Specific references are provided for recent developments.     

The use of fluorochromes in the cytogenetics of the small-grained cereals (Triticeae)

Summary This paper describes some of the major advances that have been made in the cytogenetics of the small-grained cereals (Triticeae) using fluorochromes to detect nucleic acids in situ. The method, widely known as fluorescence in situ hybridization, has made a contribution in several areas including (i) chromosome mapping programmes, and (ii) cereal breeding programmes. Flow cytometry of cereal chromosomes has now been developed for the generation of chromosome enriched libraries; these libraries will ultimately be of use in both the cereal mapping and breeding programmes. Fluorescence in situ hybridization has also made a major contribution to the understanding of cereal genome structure by elucidating the distribution of different classes of DNA sequence. By using suitable nucleic acid probes whole chromosomes can now be identified in interphase nuclei. The labelling patterns have revealed a structured arrangement of chromosomes at interphase. Not only are chromosomes organized but the ribosomal RNA genes also show structured patterns of condensation and expression. Progress in each of these areas has been rapid in recent years and this progress is described.     

Malpighian tubule polytene chromosomes of Culex quinquefasciatus (Diptera,Culicinae)

Dipteran polytene chromosomes provide an excellent model for understanding in species complexes, as well as for structural and functional cytogenetics. The status of species in the Culex pipiens complex is controversial and the use of polytene chromosomes for cytogenetic analysis in the subfamily Culicinae has been difficult because of methodological problems. In this study, Malpighian tubule polytene chromosomes were obtained from young (0 to 12 h, 20 C) and old (20 to 42 h, 28 C) laboratory-bred C. pipiens quinquefasciatus pupae. The chromosome maps for this species were constructed and compared with published data for C. pipiens pipiens and C. p. quinquefasciatus. Although the banding patterns were conserved between subspecies, analysis of the structural variations in the bands and interbands revealed differences apparently related to the physiological stage and ecogeographical strain. The organization of the centromeric regions in larval and pupal chromosomes showed greater similarity to each other than did those of pupal and adult chromosomes. The use of pupal polytene chromosomes for in situ hybridization with vector competence probes is discussed.     

18p− Syndrome: an unusual case and diagnosis by in situ hybridization with chromosome 18-specific alphoid DNA sequence

Summary A patient with an atypical clinical picture of 18p^– syndrome is described. By the in situ hybridization technique we localized the chromosome 18-specific cloned repetitive sequence to metaphase chromosomes of the patient. The predominant hybridization of the probe was found in pericentromeric regions of homologous chromosomes 18. The amount of pericentromeric DNA measured by in situ hybridization differed between homologous chromosomes; and the number of radioactive grains was statistically greater in the normal chromosome 18 than in the aberrant chromosome 18p^–. The results indicate that this probe may be useful in clinical cytogenetics for identification of aberrant chromosomes, localization of breakpoints, and studies of C-band DNA polymorphism of chromosome 18.     

Mouse satellite DNA, centromere structure, and sister chromatid pairing

The experiments described were directed toward understanding relationships between mouse satellite DNA, sister chromatid pairing, and centromere function. Electron microscopy of a large mouse L929 marker chromosome shows that each of its multiple constrictions is coincident with a site of sister chromatid contact and the presence of mouse satellite DNA. However, only one of these sites, the central one, possesses kinetochores. This observation suggests either that satellite DNA alone is not sufficient for kinetochore formation or that when one kinetochore forms, other potential sites are suppressed. In the second set of experiments, we show that highly extended chromosomes from Hoechst 33258-treated cells (Hilwig, I., and A. Gropp, 1973, Exp. Cell Res., 81:474-477) lack kinetochores. Kinetochores are not seen in Miller spreads of these chromosomes, and at least one kinetochore antigen is not associated with these chromosomes when they were subjected to immunofluorescent analysis using anti-kinetochore scleroderma serum. These data suggest that kinetochore formation at centromeric heterochromatin may require a higher order chromatin structure which is altered by Hoechst binding. Finally, when metaphase chromosomes are subjected to digestion by restriction enzymes that degrade the bulk of mouse satellite DNA, contact between sister chromatids appears to be disrupted. Electron microscopy of digested chromosomes shows that there is a significant loss of heterochromatin between the sister chromatids at paired sites. In addition, fluorescence microscopy using anti-kinetochore serum reveals a greater inter-kinetochore distance than in controls or chromosomes digested with enzymes that spare satellite. We conclude that the presence of mouse satellite DNA in these regions is necessary for maintenance of contact between the sister chromatids of mouse mitotic chromosomes.     

Classical and molecular cytogenetics of the zebrafish, Danio rerio (Cyprinidae, Cypriniformes): an overview

The zebrafish, Danio rerio, has recently become the model system for the genetic analysis of vertebrate development. This paper reviews the advances in zebrafish cytogenetics, obtained through classical and molecular techniques, which will lead to the assignment of specific linkage groups to specific chromosome pairs in the zebrafish genome project. Several chromosome pairs of the 50-chromosome karyotype of D. rerio were differentially stained by classical staining techniques and additional information has been obtained by molecular cytogenetics. Indeed, the analysis of constitutive heterochromatin by C-banding and base-specific fluorochrome staining had suggested a differential composition of peri- and paracentromeric constitutive heterochromatin. The chromosome mapping of distinct AT- and GC-rich zebrafish satellite DNAs by means of PRINS (Primed in situ) and multicolor FISH (Fluorescence in situ Hybridization) has confirmed this hypothesis, which therefore provided the chromosome localization of 10% of the zebrafish genome. The analysis of nucleolus organizer regions (NORs) by silver staining and by FISH with 18S rDNA has also revealed the existence of variable and inactive NORs, in addition to those on the terminal regions of the long arms of the three NOR-bearing chromosome pairs. Other multicopy genes, such as minor ribosomal genes, or multicopy repeats, such as telomere specific sequences, have now been mapped on zebrafish chromosomes. The latest advancement in zebrafish molecular cytogenetics is the chromosome mapping of single locus genes. Single-copy genes from each of the 25 genetic linkage groups are now being mapped on zebrafish chromosomes by using PAC clones.     

Molecular cytogenetics and taxonomy of insects,with particular reference to the coleoptera

We assess and review the impact of the new cytogenetic techniques in insects for their roles in taxonomy and for a better knowledge of their chromosomal structure. Particular emphasis is given to molecular cytogenetics by fluorescent in situ hybridization, localization of AT- or GC-rich regions with fluorochromes, and restriction endonuclease banding in beetle chromosomes. The main features of the cytogenetics of Coleoptera are treated in detail, taking into account the range of variation in chromosome number and genome size, and our in-depth findings on the constitutive heterochromatin and satellite DNAs of tenebrionid beetles. Some other topics of interest for insect cytogenetics, such as the meiotic association of sex chromosomes, the nucleolar organizing regions (NORs), and the molecular constitution of telomeres, are also discussed from the taxonomic and structural viewpoints.     

Veterinary cytogenetics: past and perspective

Cytogenetics was conceived in the late 1800s and nurtured through the early 1900s by discoveries pointing to the chromosomal basis of inheritance. The relevance of chromosomes to human health and disease was realized more than half a century later when improvements in techniques facilitated unequivocal chromosome delineation. Veterinary cytogenetics has benefited from the information generated in human cytogenetics which, in turn, owes its theoretical and technical advancement to data gathered from plants, insects and laboratory mammals. The scope of this science has moved from the structure and number of chromosomes to molecular cytogenetics for use in research or for diagnostic and prognostic purposes including comparative genomic hybridization arrays, single nucleotide polymorphism array-based karyotyping and automated systems for counting the results of standard FISH preparations. Even though the counterparts to a variety of human diseases and disorders are seen in domestic animals, clinical applications of veterinary cytogenetics will be less well exploited mainly because of the cost-driven nature of demand on diagnosis and treatment which often out-weigh emotional and sentimental attachments. An area where the potential of veterinary cytogenetics will be fully exploited is reproduction since an inherited aberration that impacts on reproductive efficiency can compromise the success achieved over the years in animal breeding. It is gratifying to note that such aberrations can now be tracked and tackled using sophisticated cytogenetic tools already commercially available for RNA expression analysis, chromatin immunoprecipitation, or comparative genomic hybridization using custom-made microarray platforms that allow the construction of microarrays that match veterinary cytogenetic needs, be it for research or for clinical applications. Judging from the technical refinements already accomplished in veterinary cytogenetics since the 1960s, it is clear that the importance of the achievements to date are bound to be matched or out-weighed by what awaits to be accomplished in the not-too-far future.     

The karyotype of Galago alleni Waterhouse 1837 (2N=40), compared with that of Galago senegalensis Geoffroy 1796 (2N=38) (primates,prosimii, Galagidae)

In a broader project on the cytogenetics of the Galagidae the chromosomal complement of a female Galago alleni was studied. The karyotype, consisting of 40 chromosomes, is described and compared with the karyotype found in three individuals of Galago senegalensis with 38 chromosomes.This study was supported by Grant No. 82-34 of the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).     

Unusual case of 18p- syndrome: diagnosis using a cloned DNA fragment

The patient with atypical clinic picture of 18p- syndrome is described. The in situ hybridization technique was used to localize chromosome 18-specific cloned sequence to metaphase chromosomes of the proband. The predominant hybridization was found in pericentromeric regions of homologous chromosome 18. The amount of pericentromeric DNA measured by in situ hybridization was different in homologous chromosomes and the number of radioactive grains was statistically greater in the normal chromosome 18 than in the chromosome 18p-. The cause of asymmetrical hybridization of probes to homologous chromosomes 18 is discussed. The results obtained indicate that this probe may be useful in clinical cytogenetics for identification of chromosome 19 in metaphase and interphase cells, determination of breakpoints or studies of pericentromeric DNA polymorphisms.     

Evolution of karyotype,sex chromosomes,and meiosis in mygalomorph spiders (Araneae: Mygalomorphae)

Spider diversity is partitioned into three primary clades, namely Mesothelae, Mygalomorphae, and Araneomorphae. Mygalomorph cytogenetics is largely unknown. Our study revealed a remarkable karyotype diversity of mygalomorphs. Unlike araneomorphs, they show no general trend towards a decrease of 2n, as the chromosome number was reduced in some lineages and increased in others. A biarmed karyotype is a symplesiomorphy of mygalomorphs and araneomorphs. Male meiosis of some mygalomorphs is achiasmatic, or includes the diffuse stage. The sex chromosome system X₁X₂0, which is supposedly ancestral in spiders, is uncommon in mygalomorphs. Many mygalomorphs exhibit more than two (and up to 13) X chromosomes in males. The evolution of X chromosomes proceeded via the duplication of chromosomes, fissions, X–X, and X‐autosome fusions. Spiders also exhibit a homomorphic sex chromosome pair. In the germline of mygalomorph males these chromosomes are often deactivated; their deactivation and pairing is initiated already at spermatogonia. Remarkably, pairing of sex chromosomes in mygalomorph females is also initiated at gonial cells. Some mygalomorphs have two sex chromosome pairs. The second pair presumably arose in early‐diverging mygalomorphs, probably via genome duplication. The unique behaviour of spider sex chromosomes in the germline may promote meiotic pairing of homologous sex chromosomes and structural differentiation of their duplicates, as well as the establishment of polyploid genomes. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 377–408.     

Chromosomal and molecular characterization of Aethomys kaiseri from Zambia and Aethomys chrysophilus from Tanzania (Rodentia, Muridae)

Aethomys is a common and widespread rodent genus in the African savannas and grasslands. However, its systematics and taxonomy are still unclear as no study has covered the entire range. In fact it might not be a monophyletic genus and perhaps should be split into two subgenera, Micaelamys and Aethomys. In this paper, we present findings based on the cytogenetics and the entire cytochrome b sequence of two species from Zambia (A. kaiseri) and Tanzania (A. chrysophilus), and we compare them with the sequences of a South African species (A. namaquensis) and other allied muroid genera. Comparison of the banded chromosomes revealed complete G-band homology between the autosomes of the two species. However, the X and Y chromosomes clearly differ in size and in C- and G-banding, being much larger in A. kaiseri. Comparison of the cytochrome b sequences places the separation between A. kaiseri and A. chrysophilus at 4.49 Mya, a period of intense speciation in other African muroids. The resulting phylogeny strongly supports the idea of a paraphyletic group, suggesting the need to elevate the previously described subgenera to the genus rank.     

Comparative evaluation of bacteria identification from the Acinetobacter genus using a commercially available API 20NE system, PCR and RFLP techniques

A study was carried out for identification of 50 Acinetobacter strains isolated from various clinical materials. Using classic methods the following species were identifies: Acinetobacter sp. (68%), Acinetobacter baumanii (24%) and Acinetobacter lwofii (8%). In all strains the recA gene was found of 435-500 pz size which confirms their belonging to that genus. Amplification products were digested with restriction enzymes Mbol and HinfI (RFLP) and their detection was carried out on agarose gel by electrophoresis methods, owing to that the arrangement of gene fragment characteristic of each strain was obtained. After careful analysis restriction patterns were obtained corresponding to the following genome species: Acinetobacter baumanii (60%), Acinetobacter sp. 3 (28%) and Acinetobacter lwoffii (12%). The methods of molecular biology made possible a more precise classification of the studied strains according to species. Certain strains determined as Acinetobacter sp. by the API 20NE system were found to be Acinetobacter baumanii, Acinetobacter sp. 3 or Acinetobacter lwofii when determined by the PCR/RFLP method.     

Comparative cytogenetic analysis between Lonchorhina aurita and Trachops cirrhosus (Chiroptera, Phyllostomidae)

Phyllostomidae comprises the most diverse family of neotropical bats, its wide range of morphological features leading to uncertainty regarding phylogenetic relationships. Seeing that cytogenetics is one of the fields capable of providing support for currently adopted classifications through the use of several markers, a comparative analysis between two Phyllostomidae species was undertaken in the present study, with a view to supplying datasets for the further establishment of Phyllostomidae evolutionary relationships. Karyotypes of Lonchorhina aurita (2n = 32; FN = 60) and Trachops cirrhosus (2n = 30; FN = 56) were analyzed by G- and C-banding, silver nitrate staining (Ag-NOR) and base-specific fluorochromes. Chromosomal data obtained for both species are in agreement with those previously described, except for X chromosome morphology in T. cirrhosus, hence indicating chromosomal geographical variation in this species. A comparison of G-banding permitted the identification of homeologies in nearly all the chromosomes. Furthermore, C-banding and Ag-NOR patterns were comparable to what has already been observed in the family. In both species CMA(3) /DA/DAPI staining revealed an R-banding-like pattern with CMA (3) , whereas DAPI showed uniform staining in all the chromosomes. Fluorochrome staining patterns for pericentromeric constitutive heterochromatin (CH) regions, as well as for nucleolar organizing regions (NORs), indicated heterogeneity regarding these sequences among Phyllostomidae species.     

A new approach in recognition of heterochromatic regions of human chromosomes by means of restriction endonucleases.

The heteromorphisms of C-band regions of human chromosomes are evaluated by means of restriction endonucleases AluI, DdeI, MboI, and RsaI. Every chromosome exhibits heteromorphic markers of the C-band regions except chromosome 8. Each enzyme was found to be highly characteristic in its staining profile, a result that clearly suggests the diversity of heterochromatin. The inherent C-band-region heterochromatin variability that is revealed by these enzymes provides a valuable tool in identifying markers as compared with other previously described techniques.     

Detection of chromosome variation in interphase by in situ hybridization with repetitive DNA probes: potential applications to cytogenetic analysis and mutagenicity testing

Individual chromosomes can be identified by means of in situ hybridization with DNA probes for chromosome-specific repetitive sequences. The efficiency and sensitivity of the method are strictly dependent on the characteristics of the probes and the experimental conditions. Using three probes with different copy numbers, we demonstrated that the target chromosomes can be visualized in interphase when the homologous sequences are repeated at least 50 times.Possible applications of interphase analysis to clinical cytogenetics and mutagenicity testing are discussed.     

Discrepancies between flow cytometric and cytogenetic studies in the detection of aneuploidy in human solid tumors

Parallel flow cytometric (FCM) cell DNA studies and cytogenetic studies were performed on clinical samples from twenty human solid tumors of various types and on cell lines established in tissue culture from three of these tumors. Six of twenty clinical samples (30%) showed concordance between flow cytometry and cytogenetics with respect to the presence or absence of aneuploidy. Among the fourteen cases with discrepancies between the two methods, 8 (40% of all cases) showed hypodiploidy by cytogenetics and had diploid DNA histograms. Three cases (15%) had prominent discrete peaks in the triploid to tetraploid region by cytogenetics but had only barely discernible corresponding peaks in the DNA histogram. In two cases (10%) cytogenetic studies revealed diffuse aneuploidy. Cytogenetic studies demonstrated near-tetraploidy in three samples, but only one of these was detected by FCM; all three cases exhibited other numerical chromosomal abnormalities. In one case aneuploidy was demonstrated by FCM and not by cytogenetics. Among the tumor cell lines established in culture, the DNA Index was often higher than the cytogenetic index. Overall, 13/20 or 65% of patients with solid tumors in this study had numerical chromosomal abnormalities that were not detected by flow cytometry. Eleven of these patients had distant metastases at the time of tumor sampling, and nine of these died of their disease within 1-11 months of the time of study.     

Induction of small-segment-translocation between wheat and rye chromosomes

A new approach to produce wheat-rye translocation, based on the genetic instability caused by monosomic addition of rye chromosome in wheat, is described. 1 283 plants from the selfed progenies of monosomic addition lines with single chromosome of inbred rye line R12 and complete chromosome complement of wheat cultivar Mianyang 11 were cytologically analyzed on a plant-by-plant basis by the improved C-banding technique. 63 of the plants, with 2n = 42, were found containing wheat-rye translocation or substitution, with a frequency of 4. 91% . Compared with the wheat parent, other 32 plants with 2n = 42 exhibited obvious phenotypic variation, but their com-ponent of rye chromosome could not be detected using the C-banding technique. In situ hybridization with a biotin-la-beled DNA probe was used to detect rye chromatin and to determine the insertion sites of rye segments in the wheat chromosomes. In 20 out of the 32 variant wheat plants, small segments of rye chromosomes were found being inserted into dif