Immunocytochemical analysis of human metaphase chromosome methylation status

The present paper describes a distribution of 5-methylcytosine-rich DNA in human metaphase chromosomes from PHA-stimulated lymphocytes. Immunocytochemical detection of 5-methylcytosine was carried out with monoclonal antibodies. Fluorescent signals were preferentially localized in certain chromosomal regions, corresponding to R-, some T-bands, pricentromeric heterochromatin, and short arms of acrocentric chromosomes. Specificity of fluorescent signals distribution along chromosomes allowed to describe a new type of human metaphase chromosomes banding pattern, which we call M-banding. Specific M-markers of landmarks were identified for each chromosome pair. The analysis of M-bands methylation status was carried out taking into account data available in literature on their nucleotide structure features, namely GC-rich H3 isochore content and CpG-islands concentration. According to our results, a high level of methylation is typical for the majority of GC-rich regions. However, certain bands of 6, 9, 10, 13, 15 chromosomes (6q15, 6q21, 6q23, 9p13, 9p22, 9p32, 10q24, 13q22, 15q15, 15q24) were shown to be hypomethylated, suggesting their special functional status in lymphocytes.     

Distribution of the tandem repeat sequences and karyotyping in cucumber (Cucumis sativus L.) by fluorescence in situ hybridization

We analyzed repeat sequences composition in the genome of cucumber inbred line 9930 using whole-genome shotgun reads. The analysis showed that satellite DNA sequences are the most dominant components in the cucumber genome. The distribution pattern of several tandem repeat sequences (Type I/II, Type III and Type IV) on cucumber chromosomes was visualized using fluorescence in situ hybridization (FISH). The FISH signals of the Type III and 45S rDNA provide useful cytogenetic markers, whose position and fluorescence intensity allow for easy identification of all somatic metaphase chromosomes. A karyotype showing the position and fluorescence intensity of several tandem repeat sequences is constructed. The establishment of this FISH-based karyotype has created the basis for the integration of molecular, genetic and cytogenetic maps in Cucumis sativus and for the ultimate genome sequencing project as well.     

Courtship and spawning sounds in bird wrasse Gomphosus varius and saddle wrasse Thalassoma duperrey

Acoustic signals from the bird wrasse Gomphosus varius and saddle wrasse Thalassoma duperrey were recorded on coral reefs in Hawaii. Terminal phase males in both species emit two types of pulse trains (type I and type II). Type I pulses were produced during spawning and courtship, while type II pulses were associated only with courtship behaviours. Gomphosus varius type I pulses were of lower frequency than T. duperrey type I pulses (271 v. 840 Hz) and were of narrower band. Discriminant function analyses revealed interspecific differences between type I pulse trains and individual pulses of both types. This study is the first documentation of courtship and spawning sounds in sympatric labrids and shows divergence in acoustic signals.     

Tomographic distribution of acetylated histone H4 in plant chromosomes,nuclei and nucleoli

Root tip cells of broad bean (Vicia faba L. cv. ’Wase soramame’) and barley (Hordeum vulgare L. cv. ’Minorimugi’) were immunostained with antibodies specific for acetylated histone H4. With an antiserum that recognizes histone H4 acetylated at lysine-5, the nucleolar organizing region (NOR) in mitotic chromosomes was strongly labeled in both species. The broad bean had two signals in the metaphase and telophase chromosome complements and four signals in the prophase and anaphase chromosome complements, while the barley had four signals in the metaphase and telophase chromosome complements and eight signals in the prophase and anaphase complements. Five different patterns of signals were observed at interphase: in type I only nucleoli were wholly stained; in type II perinucleolar knob-like signals and/or fiber-like signals emanated from the nucleus; in type III aggregate signals appeared in the nucleolus; in type IV many small dot-like signals were distributed throughout the nucleus, except nucleoli; and in type V string-like or some granule-like signals appeared in the nucleoli. Type II was very similar to previous results by in situ hybridization with sense rDNA probes. Type III was similar to the patterns of DNA synthesis recognized as chromatin domains by anti-BrdU antibodies. Type V was very similar to the results of in situ hybridization with pTa71, rDNA probes and the appearance of the dense fibrillar components of the nucleolus. Received: 7 August 1996; in revised form: 16 September 1996 / Accepted: 16 September 1996     

Satellite DNA derived from 5S rDNA in Physalaemus cuvieri (Anura, Leiuperidae)

In the present study, we describe for the first time a family of 190-bp satellite DNA related to 5S rDNA in anurans and the existence of 2 forms of 5S rDNA, type I (201 bp) and type II (690 bp). The sequences were obtained from genomic DNA of Physalaemus cuvieri from Palmeiras, State of Bahia, Brazil. Analysis of the nucleotide sequence revealed that the satellite DNA obtained by digestion with EcoRI, called PcP190EcoRI, is 70% similar to the coding region of type I 5S rDNA and 66% similar to the coding region of type II 5S rDNA. Membrane hybridization and PCR amplification of the sequence showed that PcP190EcoRI is tandemly repeated. The satellite DNA as well as type I and type II 5S rDNA were localized in P. cuvieri chromosomes by fluorescent in situ hybridization. The PcP190EcoRI sequence was found in the centromeres of chromosomes 1-5 and in the pericentromeric region of chromosome 3. Type I 5S rDNA was detected in chromosome 3, coincident with the site of PcP190EcoRI. Type II 5S rDNA was located interstitially in the long arm of chromosome 5. None of these sequences co-localized with nucleolar organizer regions. Our data suggests that this satellite DNA originates from the 5S ribosomal multigene family, probably by gene duplication, nucleotide divergence and sequence dispersion in the genome.     

Direct visualization of the sites of DNA methylation in human,and mosquito chromosomes

Human and mosquito fixed chromosomes were digested with restriction endonucleases that are inhibited by the presence of 5-methylcytosine in their restriction sites (Hha I, Hin PI, Hpa II), and with endonucleases for which cleavage is less dependent on the state of methylation (Taq I, Msp I). Methylation-dependent enzymes extracted low DNA amounts from human chromosomes, while methylation-independent enzymes extracted moderate to high amounts of DNA. After DNA demethylation with 5-azacytidine the isoschizomers Hpa II (methylation-dependent) and Msp I (methylation-independent) extracted 12-fold and 1.4-fold amounts of DNA from human chromosomes, respectively. These findings indicate that human DNA has a high concentration of Hpa II and Msp I restriction sites (CCGG), and that the internal C of this sequence is methylated in most cases, while the external cytosine is methylated less often. All the enzymes tested released moderate amounts of DNA from mosquito chromosomes whether or not the DNA was demethylated with 5-azacytidine. Hpa II induced banding in the centromere chromosome regions. After demethylation with 5-azacytidine this banding disappeared. Mosquito DNA has therefore, moderate to high frequencies of nonmethylated CpG duplets. The only exception is the centromeric DNA, in which the high levels of C methylation present produce cleavage by Hpa II and the appearance of banding. Centromere regions of human chromosomes 1 have a moderately low concentration of Hpa II-Msp I restriction sites.     

Characterization and chromosomal distribution of satellite DNA sequences of the water buffalo (Bubalus bubalis).

Satellite DNA sequences were isolated from the water buffalo (Bubalus bubalis) after digestion with two restriction endonucleases, BamHI and StuI. These satellite DNAs of the water buffalo were classified into two types by sequence analysis: one had an approximately 1,400 bp tandem repeat unit with 79% similarity to the bovine satellite I DNA; the other had an approximately 700 bp tandem repeat unit with 81% similarity to the bovine satellite II DNA. The chromosomal distribution of the satellite DNAs were examined in the river-type and the swamp-type buffaloes with direct R-banding fluorescence in situ hybridization. Both the buffalo satellite DNAs were localized to the centromeric regions of all chromosomes in the two types of buffaloes. The hybridization signals with the buffalo satellite I DNA on the acrocentric autosomes and X chromosome were much stronger than that on the biarmed autosomes and Y chromosome, which corresponded to the distribution of C-band-positive centromeric heterochromatin. This centromere-specific satellite DNA also existed in the interstitial region of the long arm of chromosome 1 of the swamp-type buffalo, which was the junction of the telomere-centromere tandem fusion that divided the karyotype in the two types of buffaloes. The intensity of the hybridization signals with buffalo satellite II DNA was almost the same over all the chromosomes, including the Y chromosome, and no additional hybridization signal was found in noncentromeric sites.     

Congenital Disorders of Glycosylation: CDG-I, CDG-II, and beyond

The Congenital Disorders of Glycosylation (CDG) are a collection of over 20 inherited diseases that impair protein N-glycosylation. The clinical appearance of CDG patients is quite diverse making it difficult for physicians to recognize them. A simple blood test of transferrin glycosylation status signals a glycosylation abnormality, but not the specific defect. An abnormal trasferrin glycosylation pattern suggests that the defect is in either genes that synthesize and add the precursor glycan (Glc(3)Man(9)GlcNAc(2)) to proteins (Type I) or genes that process the protein-bound N-glycans (Type II). Type I defects create unoccupied glycosylation sites, while Type II defects give fully occupied sites with abnormally processed glycans. These types are expected to be mutually exclusive, but a group of patients is now emerging who have variable coagulopathy and hypoglycemia together with a combination of Type I and Type II transferrin features. This surprising finding makes identifying their defects more challenging, but the defects and associated clinical manifestations of these patients suggest that the N-glycosylation pathway has some secrets left to share.     

The chromosomal organization of simple sequence repeats in wheat and rye genomes

The physical distribution of ten simple-sequence repeated DNA motifs (SSRs) was studied on chromosomes of bread wheat, rye and hexaploid triticale. Oligomers with repeated di-, tri- or tetra-nucleotide motifs were used as probes for fluorescence in situ hybridization to root-tip metaphase and anther pachytene chromosomes. All motifs showed dispersed hybridization signals of varying strengths on all chromosomes. In addition, the motifs (AG)₁₂, (CAT)₅, (AAG)₅, (GCC)₅ and, in particular, (GACA)₄ hybridized strongly to pericentromeric and multiple intercalary sites on the B genome chromosomes and on chromosome 4A of wheat, giving diagnostic patterns that resembled N-banding. In rye, all chromosomes showed strong hybridization of (GACA)₄ at many intercalary sites that did not correspond to any other known banding pattern, but allowed identification of all R genome chromosome arms. Overall, SSR hybridization signals were found in related chromosome positions independently of the motif used and showed remarkably similar distribution patterns in wheat and rye, indicating the special role of SSRs in chromosome organization as a possible ancient genomic component of the tribe Triticeae (Gramineae). Received: 13 February 1998; in revised form: 18 August 1998 / Accepted: 18 August 1998     

Mammalian adenylyl cyclase family members are randomly located on different chromosomes

Molecular cloning studies have elucidated the presence of multiple isoforms of mammalian adenylyl cyclase. So far, six different isoforms (I to VI) have been fully characterized. Comparison of their structural and biochemical characteristics suggests that the mammalian adenylyl cyclase family can be classified into four sub-families: type I, type III, type II/IV, and type V/VI. We have determined the chromosomal localization of these genes. Type I gene was assigned to chromosome 7, type III to chromosome 2, types II and IV to chromosomes 5 and 14, and types V and VI to chromosomes 3 and 12. Our results indicate that the different adenylyl cyclase isoforms, even within the same subfamily, are distributed randomly in the genome, in contrast to the chromosomal organization of other components within the same signaling pathway, such as catecholamine receptors and G proteins.     

Molecular characterization and cytogenetic analysis of highly repeated DNAs of lake trout,Salvelinus namaycush

The chromosomes of lake trout (Salvelinus namaycush) contain a considerable amount of heterochromatin located at the centromeres and/or telomeres of several chromosomes, including a sex-specific block located distally on the X chromosome. In order to investigate further the repetitive DNAs of lake trout, genomic DNA from a female was size fractionated (<600 bp) with the restriction endonuclease AluI and fragments were cloned into the bacteriophage M13. A total of 42 clones were isolated. Relative copy number of individual inserts within the lake trout genome was estimated by Southern analysis. Twelve clones were determined to be highly repetitive and were chosen for further investigation. Inserts of these clones contained sequences similar to the AluI/RsaI, EcoRI/DraI, DraI/BstEII, and MboI/BglII families reported from Arctic char (Salvelinus alpinus). The chromosomal location of several of these fragments was determined in lake trout by fluorescence in situ hybridization (FISH). Two related AluI/RsaI sequences (Type A, 140 bp, and Type B, 120 bp) showed differential hybridization. Type A hybridized to the centromeres of all metacentric as well as several acrocentric chromosomes. Type B hybridized to the centromeres of most acrocentric chromosomes. A sequence with homology to the EcoRI/DraI family hybridized to the centromeres of several acrocentric chromosomes. Sequences with partial similarity to the DraI/BstEII family hybridized to the major rDNA sites (nucleolar organizer regions, NORs) and several minor telomeric sites. The interstitial and telomeric heterochromatin of lake trout, including that of the X chromosome, appears to comprise sequences belonging to the MboI/BglII family.     

Fluorimetry of bovine myotendon junction by fibre-optics and microscopy of intact and sectioned tissues

Summary The autofluorescence of tendon, epimysium and endomysium at the myotendon junction of the deep digital flexor in the bovine forelimb was measured with a fluorescence microscope and with a bifurcated light guide composed of quartz optical fibres. Data were adjusted for spectral variation in the radiance of the halogen illuminator used to standardize the photometer. Samples of myotendon junction were examined intact, in slices several millimetres thick and after being frozen in liquid nitrogen and sectioned at 20 µm. Sections were examined with and without a mounting medium and with and without immersion oil objectives. Type I collagen fibres were identified by their scarcity of branching, relatively large size and yellow staining with silver. Type III collagen fibres were identified by their extensive branching, small size and black staining with silver. Purified Types I and III collagen were also examined. Type I collagen fibres had a strong fluorescence emission peak between 410 and 450 nm and a shoulder at 510 nm. For the strong peak, results obtained by fibre-optics were positively biased relative to those obtained by microscopy. Type III collagen reticular fibres lacked a strong emission peak at 410 to 450 nm. Although their overall fluorescence was weaker than that of Type I collagen fibres, Type III collagen fibres had similar or slightly stronger emissions around 510 nm. The Type I emission spectrum of collagen fibres was converted to a spectrum similar to the Type III spectrum by conditions that caused the fading of fluorescence (storage as dry or mounted sections and exposure of sections to UV light). It is suggested that, with fibre-optic fluorimetry of intact tissues, Type I collagen fibres may emit a pre-fading spectrum while Type III collagen fibres may emit a post-fading spectrum, and that the preservation of Type I and the fading of Type III collagen is a consequence of the surface to volume ratio of their fibres.     

Uneven extraction of protein in Chinese hamster chromosomes during G-staining procedures

To elucidate the role of chromosomal protein in G-band production, changes of protein distribution in chromosomes were studied in situ at each step of G-staining procedures. As a highly specific stain for protein, dansyl Cl was used, which conjugated with amino groups in polypeptide to emit bright fluorescence under UV irradiation, so that the pattern of fluorescence of dansyl-stained chromosomes was expected to reflect the distribution of protein. Uniform fluorescence pattern observed in untreated, dansyl-stained chromosomes indicated even distribution of protein in the ordinary air-dried chromosomes. The pattern of fluorescence representing the distribution of chromosomal protein after pretreatments of G-staining showed brighter outlines of chromatids, reduced fluorescence of chromosome body, and a slight difference in intensity along chromosome arms which corresponded to G-bands. This correspondence was confirmed when Giemsa stain was removed from G-banded chromosomes and the chromosomes were stained with dansyl Cl. The resulting dim fluorescence pattern conformed to G-bands previously observed in the same chromosomes. Similar events were observed in HCl-extracted chromosome slides, although the fluorescence was considerably reduced in this case. Our results inferred that chromosomal protein was partially lost during pretreatments of G-staining, that acid-soluble protein assumed less significant role in G-staining mechanism, and that uneven deprivation of acid-insoluble protein may occur during G-staining procedures.     

荧光原位杂交技术分析栽培种甘薯(Ipomoea batatas cv.XushuNo.18)染色体

为了解栽培种甘薯(徐薯18,Ipomoea batatas cv.XushuNo.18)的染色体结构,文章利用45SrDNA荧光原位杂交、自身基因组荧光原位杂交和银染技术对栽培种甘薯进行分子细胞遗传学研究。银染结果显示,徐薯18间期核有6对、8对和9对银染点;45SrDNA荧光原位杂交结果显示,徐薯18染色体上有8对或9对强弱不一的45SrDNA信号;自身基因组荧光原位杂交结果表明,所有染色体的全长分布强烈而密集的杂交信号,着丝粒区、近着丝粒区和端粒区有增强的信号带。     

Localization of the ribosomal genes in Caenorhabditis elegans chromosomes by in situ hybridization using biotin-labeled probes

The site of the ribosomal gene cluster on embryonic metaphase chromosomes of Caenorhabditis elegans has been mapped by in situ hybridization using probe DNAs that have been nick-translated to incorporate biotin-labeled UTP. The hybridized probe DNA was detected by a double-layer fluorescent antibody technique. Since chromosomes from wild-type C. elegans embryos are indistinguishable, in situ hybridization was carried out with chromosomes from C. elegans strains carrying cytologically distinct translocation or duplication chromosomes in order to identify the right end of linkage group I as the site of the ribosomal genes. Chromosomes carrying a lethal mutation, let-209 I displayed smaller hybridization signals than wild-type, suggesting that these chromosomes carried a partial deficiency of the ribosomal gene cluster. A duplication of the ribosomal genes, eDp20(I;II) rescued let-209 homozygotes. Chromosomes carrying the alterations in the ribosomal genes were combined with mnT12(IV;X) to facilitate the mapping of genes in C. elegans by in situ hybridization. Linkage groups I and II are then labeled by the distinctive hybridization signals from the ribosomal probes, linkage groups IV and X are together distinguishable morphologically and linkage group V is labeled by hybridization to a 5S gene probe.     

Sex chromosome linkage of chicken and duck type I interferon genes: further evidence of evolutionary conservation of the Z chromosome in birds

Type I interferons (IFNs) are a family of proteins that are predominantly expressed in response to viral infection. Two serologically distinct forms of type I IFN, designated ChIFN1 and ChIFN2, have recently been recognized in the chicken. ChIFN1 is encoded by a cluster of ten or more intronless genes, whereas ChIFN2, whose primary sequence is 57% identical, is encoded by a single intronless gene. By fluorescence in situ hybridization we now demonstrate that the genes for ChIFN1 and ChIFN2 are all located on the short arm of the chicken Z chromosome. This assignment was confirmed by results that showed that DNA from male (ZZ) chickens yielded approximately twofold stronger Southern blot signals with ChIFN1 and ChIFN2 hybridization probes than DNA from females (ZW). Attempts to determine differences in IFN production between male and female chickens failed owing to a high degree of variation in virus-induced IFN expression between individuals of both sexes. Sex linkage of IFN genes was also observed in domestic ducks: fluorescence in situ hybridization of duck metaphase chromosomes with a duck type I IFN probe was confined to the terminal region of the long arm of the Z chromosome. Thus, in contrast to mammals, which have their IFN genes on autosomes, birds have the type I IFN genes on the sex chromosome. Received: 9 January 1998     

High-resolution structural analysis of biolistic transgene integration into the genome of wheat

Transformation of plant genomes by biolistic methods has become routine over the past decade. However, relatively little is known about how transgenes are physically integrated into the host genome. Using a high-resolution physical mapping technique, fluorescence in situ hybridization on extended DNA fibers (fiber-FISH), 13 independent transgenic wheat lines were analyzed to determine the structural arrangement of stably inherited transgenes in host-plant chromosomes. Twelve transgenic lines were transformed with a single plasmid and one line was co-transformed with two separate plasmids, which co-segregated genetically. Three basic integration patterns were observed from the fiber-FISH experiments: Type I, large tandemly repeated integration; Type II, large tandem integrations interspersed with unknown DNA; and Type III, small insertions, possibly interspersed with unknown DNA. Metaphase FISH showed that the integration of transgenes was in both hetero- and euchromatic, as well as proximal, interstitial and distal, regions of the chromosomes. In the transgenic plants, the type of promotor used, rather than the chromosomal site of transgene integration, was most critical for transgene expression. The integration of the transgenes was not associated with detectable chromosomal rearrangements. Received: 25 August 2000 / Accepted: 31 October 2000     

FISH分析栽培高粱×拟高粱、甜高粱×拟高粱的染色体行为

采用荧光原位杂交技术对45S rDNA在栽培高粱×拟高粱、甜高粱×拟高粱F1的有丝分裂和减数分裂染色体进行定位研究。在有丝分裂中期染色体上2个杂种分别检测到2个杂交信号, 在减数分裂粗线期、终变期、中期Ⅰ染色体上45S rDNA位于一个二价体上, 说明这两个杂种携带45S rDNA的染色体为同源染色体。根据45S rDNA位点随细胞减数分裂过程的位置变化, 表明这两个杂种染色体配对行为正常, 平均构型为2n=2x=20(10Ⅱ), 证明45S rDNA可作为染色体的一个识别指标间接地观察细胞减数分裂过程染色体的变化行为。