Preferential expression of unique sequences adjacent to middle repetitive sequences in mouse cytoplasmic RNA.

Total single-copy DNA and single-copy DNA contiguous to middle repetitive sequences were isolated from mouse brain by successive hydroxylapatite column chromatographies. These DNAs, termed repeat-contiguous single-copy DNA, were found to constitute 48% of the total single-copy DNA. The saturation hybridization values of these two DNA probes to nuclear RNA and cytoplasmic RNA containing polyA of mouse brain and liver were measured. The saturation hybridization levels of total single-copy DNA to brain and liver nuclear RNA were 13.5% and 8.8%, respectively, and those of repeat-contiguous single-copy DNA to the same RNA samples were 13.3% and 8.5%, respectively. On the contrary, the saturation hybridization levels of single-copy DNA to cytoplasmic RNA containing polyA of brain and liver were 3.8% and 2.0%, respectively, and those of repeat-contiguous single-copy DNA to the same RNA samples were 5.8% and 4.0%, respectively. Similar results were obtained with total cytoplasmic RNA. These results indicate that about half the steady state nuclear RNA is transcribed from repeat-contiguous single-copy DNA, and that cytoplasmic RNA containing polyA is mainly derived from repeat-contiguous single-copy DNA.     

Prophase chromosome unique band sequences: definition and utilization

Extensive experience with the analysis of human prophase chromosomes and studies into the complexity of prophase banding patterns have suggested that at least some prophase chromosomal segments can be accurately identified and characterized independently of the morphology of the chromosome as a whole. The feasibility of identifying and analyzing specified prophase chromosome segments was thus investigated as an alternative approach to prophase chromosome analysis based on whole-chromosome recognition. Through the use of prophase idiograms at the 850-band stage (Francke, 1981) and a systematic comparison system, we have demonstrated that it is possible to divide the 24 human prophase idiograms into a set of 94 unique band sequences, each of which has a banding pattern that is recognizable and distinct from any other nonhomologous chromosome portion. The use of a unique band sequence approach in prophase chromosome analysis is expected to increase efficiency and sensitivity through more effective use of available banding information.     

Intracellular localization and unique conserved sequences of three small nucleolar RNAs.

Three human small nucleolar RNAs (snoRNAs), E1, E2 and E3, were reported earlier that have unique sequences, interact directly with unique segments of pre-rRNA in vivo and are encoded in introns of protein genes. In the present report, human and frog E1, E2 and E3 RNAs injected into the cytoplasm of frog oocytes migrated to the nucleus and specifically to the nucleolus. This indicates that the nucleolar and nuclear localization signals of these snoRNAs reside within their evolutionarily conserved segments. Homologs of these snoRNAs from several vertebrates were sequenced and this information was used to develop RNA secondary structure models. These snoRNAs have unique phylogenetically conserved sequences.     

A unique carbohydrate binding domain targets the lafora disease phosphatase to glycogen.

Lafora disease (progressive myoclonus epilepsy of Lafora type) is an autosomal recessive neurodegenerative disorder resulting from defects in the EPM2A gene. EPM2A encodes a 331-amino acid protein containing a carboxyl-terminal phosphatase catalytic domain. We demonstrate that the EPM2A gene product also contains an amino-terminal carbohydrate binding domain (CBD) and that the CBD is critical for association with glycogen both in vitro and in vivo. The CBD domain localizes the phosphatase to specific subcellular compartments that correspond to the expression pattern of glycogen processing enzyme, glycogen synthase. Mutations in the CBD result in mis-localization of the phosphatase and thereby suggest that the CBD targets laforin to intracellular glycogen particles where it is likely to function. Thus naturally occurring mutations within the CBD of laforin likely result in progressive myoclonus epilepsy due to mis-localization of phosphatase expression.     

Identification of associating carbohydrate sequences with labelled oligosaccharides

The gelling subunit of alginate, the major cell-wall polysaccharide of brown algae, was used as a molecular marker for identification of this cell-wall carbohydrate subunit at the cellular level. Short polyguluronate chains were conjugated to fluorescein and used as a probe to identify the gelling regions of alginate in tissue sections from a brown alga. The specificity of the probe for gelling subunits was demonstrated by lack of cell-wall labelling in the absence of calcium, correlation between divalent-cation binding affinities of polyguluronate with labelling intensity, and lack of labelling by fluorescein-conjugated nongelling subunits. The probe labelling-pattern also differed from sulfated fucan distribution. Extracellular matrix and cell walls were labelled on sections of vegetative blade, stipe and reproductive frond of Fucus gardneri Silva. Probe labelling was rapid, being virtually complete within 5 min. Probe labelling in seawater differed markedly from labelling at lower ionic strength and is interpreted as reflecting alginategelling properties in natural conditions. High-and low-affinity binding sites are discussed in terms of gelling-subunit length and steric availability. Fluorescein-conjugated polygalacturonate, which also forms calcium dimers, labelled extracellular alginate by formation of mixed polygalacturonate-polyguluronate dimers. Binding by the alginate hybridization probe differs from nucleic-acid hybridization in divalent-cation bridging and the lack of both a conformational transition and polymer polarity.Abbreviations EDTA ethylenedinitrilo tetraacetic acid (ethylenediaminetraacetic acid) - NMR nuclear magnetic resonance spectroscopy     

Evolution of four human Y chromosomal unique sequences

Four cloned unique sequences from the human Y chromosome, two of which are found only on the Y chromosome and two of which are on both the X and Y chromosomes, were hybridized to restriction enzyme-treated DNA samples of a male and a female chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), and pig-tailed macaque (Macaca nemestrina); and a male orangutan (Pongo pygmaeus) and gibbon (Hylobates lar). One of the human Y-specific probes hybridized only to male DNA among the humans and great apes, and thus its Y linkage and sequence similarities are conserved. The other human Y-specific clone hybridized to male and female DNA from the humans, great apes, and gibbon, indicating its presence on the X chromosome or autosomes. Two human sequences present on both the X and Y chromosomes also demonstrated conservation as indicated by hybridization to genomic DNAs of distantly related species and by partial conservation of restriction enzyme sites. Although conservation of Y linkage can only be demonstrated for one of these four sequences, these results suggest that Y-chromosomal unique sequence genes do not diverge markedly more rapidly than unique sequences located on other chromosomes. However, this sequence conservation may in part be due to evolution while part of other chromosomes.     

Efficient capture of unique sequences from eukaryotic genomes

Cot-based cloning and sequencing (CBCS), a synthesis of Cot analysis, DNA cloning and high-throughput sequencing, promises to accelerate the study of eukaryotic genomes. In particular, CBCS will (1) permit efficient gene discovery in species with substantial quantities of repetitive DNA, (2) allow the sequence complexity (i.e. all the unique sequence information) of large genomes to be elucidated at a fraction of the cost of shotgun sequencing, and (3) enhance genome sequencing efforts by facilitating capture of low-copy sequences not secured by EST sequencing. CBCS should accelerate comparative genomics research, especially in large genomes such as those of many crops.     

Isolation and regional mapping of DNA sequences unique to human chromosome 21.

To isolate DNA sequences unique to chromosome 21 we have used a recombinant-DNA library, constructed from a mouse-human somatic-cell hybrid line containing chromosome 21 as the only human chromosome. Individual recombinant phage containing human DNA inserts were identified by their hybridization to total human DNA sequences and by their failure to hybridize to total mouse DNA sequences. A repeat-free human DNA fragment was then subcloned from each of 14 such recombinant phage. An independent somatic-cell hybrid was used to assign all 14 subcloned fragments to chromosome 21. Thirteen of the fragments have been regionally mapped using a somatic-cell hybrid containing a human 21 translocation chromosome. Two probes map proximal to the 21q21.2 translocation breakpoint, and 11 probes map distal to this breakpoint, placing them in the region 21q21.2-21q22. One of seven probes used to screen for restriction-fragment-length polymorphisms recognized polymorphic DNA fragments when hybridized to genomic DNA from unrelated individuals. These 14 unique probes provide useful tools for studying the structure and function of human chromosome 21 as well as for investigating the molecular biology of Down syndrome.     

Conservation and characterization of unique porcine interstitial telomeric sequences

Telomeres are composed of TTAGGG repeats and located at the ends of chromosomes. Telomeres protect chromosomes from instability in mammals, including mice and humans. Repetitive TTAGGG sequences are also found at intrachromosomal sites, where they are named as interstitial telomeric sequences (ITSs). Aberrant ITSs are implicated in chromosomal instability and found in cancer cells. Interestingly, in pigs, vertebrate telomere sequences TTAGGG (vITSs) are also localized at the centromeric region of chromosome 6, in addition to the end of all chromosomes. Surprisingly, we found that botanic telomere sequences, TTTAGGG (bITSs), also localize with vITSs at the centromeric regions of pig chromosome 6 using telomere fluorescence in situ hybridization (FISH) and by comparisons between several species. Furthermore, the average lengths of vITSs are highly correlated with those of the terminal telomeres (TTS). Also, pig ITSs show a high incidence of telomere doublets, suggesting that pig ITSs might be unstable and dynamic. Together, our results show that pig cells maintain the conserved telomere sequences that are found at the ITSs from of plants and other vertebrates. Further understanding of the function and regulation of pig ITSs may provide new clues for evolution and chromosomal instability.     

The use of subchromosome-length unique band sequences in the analysis of prophase chromosomes.

Using human prophase chromosome ideograms at the 850-band stage, we previously demonstrated that the 24 prophase ideograms can be divided into a set of 94 unique band sequences, each having a recognizable banding pattern distinct from other nonhomologous chromosome portions. Using actual prophase mitotic cells in this study, we analyzed the p arm of chromosome 11 and of chromosomes 16-22 and characterized a similar set of unique band sequences on actual chromosomes. This set of unique band sequences, a statistical comparison scheme, and image-processing techniques outlined in the present report can be used to identify and distinguish banding patterns of these chromosomes and to determine band pattern abnormalities.