Sex specific chromosome polymorphisms in the common Indian Krait,Bungarus caeruleus Schneider (Ophidia,Elapidae)

Chromosome analyses of common Indian Krait, B. caeuleus from three geographical regions of India have revealed variable diploid numbers of 43, 44 and 45 in different female individuals but a constant diploid number of 44 in the males. C-banding and in situ hybridization studies, using radio labelled W sex chromosome specific satellite DNA as a probe, have shown that C-banding and sex chromosome associated satellite DNA's are exclusively localised in the W chromosome. The W chromosome is involved in reciprocal translocations either with a medium sized macroautosome or with a microchromosome resulting in a multiple sex chromosome constitution of Z₁Z₁Z₂Z₂/Z₁Z₂W type. In some female individuals dissociation of the W has resulted in multiple W chromosomes, W₁ and W₂. These polymorphisms are uniquely confined to the female sex only. A predominance of polymorphic females, involving particularly the translocation of a medium sized macrochromosome, in all three geeographical regions and the restriction of the females having the original chromosome constitution (ZW) to one geographical region suggests that polymorphic individuals have adaptive flexibility and higher fecundity.     

Isolation and characterization of an α-satellite repeated sequence from human chromosome 22

We constructed a library in IL47.1 with DNA isolated from flow-sorted human chromosome 22. Over 50% of the recombinants contained the same highly repetitive sequence. When this sequence was used to probe Southern blots of EcoRI-digested genomic DNA, a ladder of bands with increments of about 170 bp was observed. This sequence comigrates with satellite III in Ag⁺/Cs₂SO₄ gradients and may account for at least part of the 170 bp Hae III ladder seen in isolated satellite III DNA. Partial sequence analysis revealed homology to the 171 bp monomeric repeat unit of -R1-DNA and the X specific -satellite consensus sequence. After low stringency in situ hybridization, silver grains were found over the centromeres of a number of chromosomes. Under high stringency conditions, however, the labeling was concentrated over the centromeric region of chromosome 22. This localization was confirmed using DNA from a panel of human/hamster cell lines which showed that the homologous 2.1 and 2.8 kb EcoR1 restriction fragments were chromosome 22 specific. These clones therefore contain chromosome 22 derived -satellite sequences analogous to other chromosome-specific satellite sequences described previously.     

Fluorescence in situ hybridization reveals a break in the α-satellite DNA of chromosome 1 in a family with a balanced whole-arm translocation

We have characterized a whole-arm translocation involving chromosomes 1 and 19 by traditional cytogenetic methods and fluorescence in situ hybridization with chromosome-specific -satellite and whole-chromosome painting probes, and different satellite III DNA probes. We have identified a break in the -satellite DNA region of chromosome 1, with division of this material into two a-satellite DNA blocks. This leaves one translocation chromosome with truncated -satellite DNA from chromosome 1 and the other trranslocation chromosome with all the -satellite DNA from chromosome 19 and truncated -satellite DNA from chromosome 1. We speculate whether the recombination event observed has taken place in tetraplex structures of satellite III DNA interspersed between -satellite DNA.     

Locations of a Human Satellite DNA in Human Chromosomes

USING techniques for DNA/RNA or DNA/DNA hybridization in situ, Pardue and Gall¹ and Jones² made several significant discoveries on the chromosomal locations of the mouse satellite DNA: (1) this fraction of DNA is found in all chromosomes except the Y, (2) the cytological location of the satellite DNA is limited to the centromeric region of each chromosome and is probably absent in other regions and (3) the centromeric regions of all mouse chromosomes are hetero-chromatic.     

Species-specific DNA sequences for identification of somatic hybrids between Lycopersicon esculentum and Solanum acaule

Summary Species-specific highly repeated DNA sequences can be used to screen the progeny of protoplast fusions combining different species. Such probes are easy to clone and can be detected by fast methods, e.g., hybridization to total genomic DNA. Furthermore, due to their high copy number, hybridization signals are strong and represent more than one locus, unlike isozymes or resistance markers. After cloning and screening for species-specific DNA sequences we characterized the highly repeated DNA sequences of the solanaceous species Solanum acaule and Lycopersicon esculentum var. gilva. DNA sequencing and hy ridization revealed a prominent, tandemly arranged satellite DNA repeat of 162 bp in Lycopersicon esculentum and a different satellite repeat of 183 bp, also tandemly organized, in Solanum acaule. Each repeat is absent in the respective other species. Therefore, we have used these DNA repeats as markers to distinguish regenerated interspecific somatic hybrids from the respective fusion partners. These hybrids were clearly identified by Southern hybridization and dot-blot assays to the respective ³²P-labelled satellite DNA.     

Multicolor fluorescence in situ hybridization and pulsed field electrophoresis dissect CMT1B gene region

Summary We have used multicolor fluoresence in situ hybridization of banded chromosomes to orient FcRII and clone 1054 on a single early metaphase chromosome band (1q22) representing about 2% of the physical map of chromosome 1 in the Charcot-Marie-Tooth (CMT1B) gene region. These two cloned fragments are on the same partially digested 900-kb MluI fragment detected by pulsed field gel electrophoresis. When applied to data from an earlier study, multicolor in situ hybridization results further refined the CMT1B genetic location from an 18 cM interval to a 6 cM interval and the physical map from 15% of chromosome 1 to 3% of chromosome 1. Occasionally the three FcRII immunoglobulin receptor genes within the 200-kb region are resolved in individual metaphase chromatids.     

Control of DNA replication and spatial distribution of defined DNA sequences in salivary gland cells of Drosophila melanogaster

In dividing cells, each sequence replicates exactly once in each S-phase, but in cells with polytene chromosomes, some sequences may replicate more than once or fail to replicate during S-phase. Because of this differential replication, the control of replication in polytene cells must have some unusual features. Dennhöfer (1982a) has recently concluded that the total DNA content of the polytene cells of Drosophila salivary glands exactly doubles in each S-phase. This observation, along with previous studies demonstrating satellite underreplication in salivary gland cells, led us to consider the hypothesis that there is a doubling of DNA mechanism for the control of DNA replication in polytene cells. With this mechanism, a doubling of DNA content, rather than the replication of each sequence, would signal the end of a cycle of DNA replication. To test this hypothesis, we have reinvestigated the replication of several sequences (satellite, ribosomal, histone and telomere) in salivary gland cells using quantitative in situ hybridization. We find that underreplication of some sequences does occur. In addition we have repeated Dennhöfer's cytophotometric and labeling studies. In contrast to Dennhöfer, we find that the total DNA contents of nonreplicating nuclei do reflect this partial replication, in accord with Rudkin's (1969) result. We conclude that DNA replication in polytene cells is controlled by modifications of the mechanism operating in dividing cells, where control is sequence autonomous, and not by a doubling of DNA mechanism. — In situ hybridization to unbroken salivary gland nuclei reveals the distribution of specific sequences. As expected, satellite, histone and 5S sequences are usually in a single cluster. This rules out the possibility that sequences known to be underreplicated in chromosomal DNA exist as extrachromosomal copies. Telomere sequences are grouped into two to six clusters, as if the chromosome ends are partially but not completely paired in salivary gland nuclei.     

Gene mapping from a bovine 1;29 DNA library prepared with chromosome microdissection

Bovine gene mapping is progressing rapidly using syntenic group mapping based on somatic cell hybrids and linkage, and to a lesser extent on in situ hybridization. Single chromosome DNA libraries are a logical next step, and this was, therefore, the aim of our laboratory. Since we have access to several cattle with t(1;29) and this chromosome is readily distinguishable, we chose this as our first target—recognizing that we would not produce a single chromosome library in the strict sense because two autosomes are represented. We utilized an inverted microscope and a micromanipulator fitted with glass instruments pulled specifically to dissect off approximately 100 t(1:29) chromosomes per microdrop. A glass chamber made to accommodate a hanging drop was used to extract the DNA under a dissecting microscope. The DNA was then cleaved with EcoRI and inserted in gtwes arms. Host cells were then infected with these phage and positive clones obtained. The first clone, isolated from this library by hybridization with a human collagen 6A1 cDNA, was mapped by in situ hybridization to bovine Chromosome (Chr) 1q12–q14, near the centromere. The second clone, an anonymous DNA fragment (D1S11), was mapped to 1q43–q46, near the terminal end.     

Molecular gene mapping of human aldolase A (ALDOA) gene to chromosome 16

Summary Mapping of human aldolase A (ALDOA) gene was performed by molecular hybridization techniques using a panel of human-mouse cell hybrids and sorted fractions of human metaphase chromosomes besides in situ hybridization. For the purpose, three kinds of DNA probes derived from the coding region (probe-1), the 3 noncoding region (probe-2), and the coding and 3 noncoding regions (probe-3) of human aldolase A cDNA clone, pHAAL116-3, were selectively employed. The results of RNA and DNA blot analyses indicated that the human ALDOA gene is located on chromosome 16. The in situ hybridization experiment also indicated that the ALDOA gene was localized to 16q22–q24.     

Conservation and chromosomal localization of DNA satellites in balenopterid whales

DNA satellites were isolated from three balenopterid species, viz. the minke, sei, and fin whales. In each of them at least two DNA satellites were recognizable with buoyant densities in neutral CsCl of =1.702/1.703 and =1.710/1.711, respectively. cRNAs from each satellite group were used for filter and in situ hybridisations. Homo- and heterologous DNA-cRNA hybrids within each satellite group yielded virtually identical melting curve profiles showing conservation of at least a considerable part of the DNA satellite sequences. There was no evident sequence homology between the =1.702/1.703 and the =1.710/1.711 satellites by filter hybridisation. — The in situ hybridisation showed that in each species the =1.702/1.703 satellite was located in centromeric-paracentromeric C-bands in a few pairs, whereas the =1.710/1.711 satellite was located in terminal C-bands throughout the karyotypes. — The data on the whale DNA satellites indicate that the quantitative evolution of the satellite DNA sequences preceded species divergence of the balenopterids and that the satellite sequences have remained relatively unaltered since the divergence took place. The function of satellite DNA is considered to imply the introduction of both chromosomal and genic polymorphisms and thus being of great importance in speciation. Based upon these concepts a model is postulated for the function of satellite DNA. According to this model at meiotic pairing euchromatin-heterochromatin overlapping between homologous chromosomes is considered to be of a general occurrence. This overlapping is presumed to be accentuated by the size heteromorphism frequently observed between homologous heterochromatic segments (C-bands). In the region of such euchromatin-heterochromatin overlapping, crossing-over would be excluded. The overlapping is suggested to be rectified progressively in the chromosome arms, leaving unaffected crossing-over distant to the euchromatin-heterochromatin junctions. The consequence of this will be that genes in the proximity of the junctions are collectively inherited and selected, whereas genes distant to the heterochromatin will be independently assorted and selected.     

Organization and genomic distribution of “82H” alpha satellite DNA

Summary We have investigated the organization and genomic distribution of sequences homologous to p82H, a cloned human alpha satellite sequence purported, based on previous in situ hybridization experiments, to exist at the centromere of each human chromosome. We report here that, using Southern blotting analysis under conditions of high stringency, p82H hybridizes solely to a low-copy or single-copy alphoid domain located at or near the centromeric region of human chromosome 14. In contrast, conditions of reduced hybridization stringency permit extensive cross-hybridization with nonidentical, chromosome-specific alpha satellite subsets found elsewhere in the human genome. Thus, the previously described ubiquity of 82H human centromeric sequences reflects the existence of diverse alpha satellite subsets located at the centromeric region of each human chromosome.     

Molecular cytogenetics of the equidae

A (G + C)-rich density satellite DNA ( = 1.713 gm/cc) has been purified from splenic DNA of Przewalski's horse, Equus przewalskii, by successive equilibrium density gradient centrifugations. The purified satellite, which may comprise as much as 29% of the total DNA, renatures rapidly; however, analyses of native, single-stranded, and reassociated molecules by analytical ultracentrifugation and melting properties suggests that some sequence heterogeniety exists in the 1.713 gm/cc satellite. Complementary RNA (cRNA) transcribed from the satellite DNA has been utilized for in situ hybridization studies with E. przewalskii metaphase chromosomes previously identified by quinacrine-banding. These studies establish that sequences complementary to the 1.713 g/cc satellite are greatly enriched in the centromeres of some, but not all, chromosomes. The differential distribution of satellite DNA sequences over heterochromatic regions allows discrimination of three classes of heterochromatin and serves to define three types of pericentromeric regions in the karyotype of this endangered equine species. Additionally, apparent polymorphism in concentrations of satellite DNA sequences between homologs in the same karyotype is noted.     

Meiotic chromosome behaviour reflects levels of sequence divergence inSus scrofa domestica satellite DNA

We present a general model for the evolution of chromosome-specific satellite DNA subfamilies.Sus scrofa domestica has a bimodal karyotype with two autosomal subsets of 12 meta-/submetacentric (Mc) and 6 acrocentric (Ac) chromosome types (Mc and Ac subgenomes). We show that the centromeric heterochromatin is characterised by two distinct satellite DNA families designed Mc1 and Ac2. Mc1 is a diverse satellite family of the Mc subgenome of which certain members with a 100 bp repeat unit are found to occur at the pericentromeric regions of each Mc autosome, while others are chromosome-specific, e.g. clone Mc pAv1.5, a higher order repeat variant, which hybridises specifically to chromosome 1. Ac2 is a homogeneous satellite occurring at the subterminal pericentromeric regions of all Ac autosomes. DNA sequence analyses showed that all clones investigated are built up from a 14 bp repeat unit which is highly conserved. In situ hybridisation to meiotic pachytene nuclei revealed a distinct spatial arrangement of the Ac2 centromeric satellite.     

A Chromosome 4 Satellite I DNA Isolated from SV40-Transformed Human Cells

Analysis of cellular DNA insert isolated from a free replicativeplasmid rescued from human cells transformed with an SV40 vectorplasmid revealed the presence of two arrays of repetitive DNAarranged in tandem. One sequence was homologous to the consensussequence of the human satellite DNA and the adjoining sequencewas a satellite DNA sequence which consisted of repetitive unitsof 42 base pairs (bp) and was designated HR42. The degree ofhomology between repetitive units was about 92%. By Southernanalysis the HR42 sequence was detected in HHW416, a somaticcell hybrid containing human chromosome 4, but not in HDm-5,the somatic cell hybrid which has human chromosome 14. By fluorescencein situ hybridization this repetitive DNA was assigned uniquelyto the centromeric region of human chromosome 4. These resultsshow that HR42 belongs to a subfamily of satellite I DNA specificfor human chromosome 4.     

Localization of plasmidlike DNA in giant-celled marine green algae

Summary Novel extrachromosomal DNA molecules were localized in giant-celled marine green algae by organelle isolation and fluorescence in situ hybridization methodologies. Nucleic acids extracted from isolated chloroplasts ofErnodesmis verticillata andVentricaria ventricosa were greatly enriched in plasmidlike DNA species. Fluorescence in situ hybridization was employed to resolve further the subcellular location of these molecules. Cloned restriction fragments of the algal plasmidlike DNA hybridized solely to low-molecular-weight DNA in Southern blots; they did not hybridize to any chromosomal DNA. Probes were generated from these clones that either did (Northern-positive) or did not (Northern-negative) hybridize to RNA species in Northern blots. Probes specific for localizing the plasmidlike DNA were generated from the latter clones, whereas probes potentially localizing both DNA and relevant mRNA species were generated from the former ones. After hybridization and signal amplification via indirect immunofluorescence, fluorescent punctae were visible surrounding the single pyrenoid in each chloroplast with both types of probes. The punctae were arranged in a hollow spherical configuration, as resolved by confocal laser scanning microscopy. Nearly twice as many punctae per chloroplast were present inV. ventricosa (11.5) as there were inE. verticillata (6.0). The differential distribution of plasmidlike DNA within each chloroplast was in contrast to chloroplast chromosomal DNA, which occurred as multiple nucleoids scattered throughout the entire organelle. The localization of plasmidlike DNA within chloroplasts correlates well with previous sequence data indicating that these molecules contain putative open reading frames encoding protein components of photosystems I and II.Abbreviations CLSM confocal laser scanning microscopy - DAPI 4,6-diamidino-2-phenylmdole - FITC fluorescein isothiocyanate - FISH fluorescence in situ hybridization - HMW high molecular weight - LMW low molecular weight - ORF open reading frame     

Satellite DNA and cytological staining patterns in heterochromatic inversions of human chromosome 9

Summary A series of partial inversions of the heterochromatic C-band of chromosome 9 have been stained with distamycin A plus 4,6-diamidino-2-phenyl-indol-2 HCl (DA/DAPI) and found to consist of three classes: (a) those in which only the C-band in the long arm fluoresces with DA/DAPI (these are the most frequent), (b) those in which only the C-band in the short arm fluoresces with DA/DAPI, and (c) those in which the C-bands in both arms fluoresce with DA/DAPI.There are also differences in the satellite DNA content of each type of inversion as measured by hybridisation in situ. Types (a) and (b) have satellite DNA contents similar to those of their normal homologues, while type (c) has a satellite DNA content almost double that of the normal homologue.It appears that DA/DAPI specifically stains heterochromatin that contains satellite DNA.The ability to distinguish these three types of inversion may help to resolve the question of the clinical significance of such inversions.     

Satellite DNA sequences in the human acrocentric chromosomes: information from translocations and heteromorphisms.

Satellite III DNA has been located by in situ hybridization in chromosomes 1, 3--5, 7, 9, 10, 13--18, 20--22, and Y and ribosomal DNA (rDNA) in the acrocentric chromosomes 13--15, 21, and 22. In the acrocentric chromosomes, the satellite DNA is located in the short arm. Here we report comparisons by in situ hybridization of the amount of satellite DNA in Robertsonian translocation and "normal variant" chromosomes with that in their homologs. In almost all dicentric Robertsonian translocations, the amount of satellite DNA is less than that in the normal homologs, but it is rarely completely absent, indicating that satellite DNA is located between the centromere and the nucleolus organizer region (NOR) and that the breakpoints are within the satellite DNA. The amount of satellite DNA shows a range of variation in "normal" chromosomes, and this is still more extreme in "normal variant" chromosomes, those with large short arm (p+ or ph+) generally having more satellite DNA than those with small short arms (p- or ph-). The cytological satellites are heterogeneous in DNA content; some contain satellite DNA, others apparently do not, and the satellite DNA content is not related to the size or intensity of fluorescence of the satellites. The significance of these variations for the putative functions of satellite DNA is discussed.     

On the origin of lateral asymmetry

Lateral asymmetry refers to unequal fluorescent intensity between adjacent regions of sister chromatids. It has been observed in the centromeric regions of mitotic chromosomes of mouse or human origin when cells are grown in 5-bromo-2-deoxyuridine (BrdU) for a single round of DNA synthesis. The chromosome-orientation fluorescence in situ hybridization (CO-FISH) technique was used with pseudodiploid mouse cells to show that the regions of asymmetrical brightness coincide with major satellite repetitive DNA, and that the more heavily BrdU-substituted chromatid is the one that fluoresces less brightly. These observations support a 20 year old hypothesis on the origin of lateral asymmetry. Other observations suggest that differential loss of DNA from the heavily substituted chromatid also contributes to lateral asymmetry.     

Karyotypic evolution of a novel cervid satellite DNA family isolated by microdissection from the Indian muntjac Y-chromosome

A minilibrary was constructed from DOP-PCR products using microdissected Y-chromosomes of Indian muntjac as DNA templates. Two microclones designated as IM-Y4-52 and IM-Y5-7 were obtained from negative screening of all three cervid satellite DNAs (satellites I, II, and IV). These two microclones were 295 and 382 bp in size, respectively, and shared 70% sequence homology. Southern blot analysis showed that the IM-Y4-52 clone was repetitive in nature with an 0.32-kb register in HaeIII digest. Sequence comparison revealed no similarities to DNA sequences deposited in the GenBank database, suggesting that the microclone sequences were from a novel satellite DNA family designated as cervid satellite V. A subclone of an Indian muntjac BAC clone which screened positive for IM-Y4-52 had a 3,325-bp insert containing six intact monomers, four deleted monomers, and two partial monomers. The consensus sequence of the monomer was 328 bp in length and shared more than 80% sequence homology with every intact monomer. A zoo blot study using IM-Y4-52 as a probe showed that the strong hybridization with EcoRI digested male genomic DNA of Indian muntjac, Formosan muntjac, Chinese muntjac, sambar deer, and Chinese water deer. Female genomic DNA of Indian muntjac, Chinese water deer, and Formosan muntjac also showed positive hybridization patterns. Satellite V was found to specifically localize to the Y heterochromatin region of the muntjacs, sambar deer, and Chinese water deer and to chromosome 3 of Indian muntjac and the X-chromosome of Chinese water deer.Y.-C. Li and Y.-M. Cheng contributed equally to this work.