Deoxyribonucleic acid of the crab Cancer pagurus : III. Intracellular localization of poly d(A---T) of Cancer pagurus by hybridization in situ

The satellite DNA poly [d(A---T) · d(T---A)] of the crab Cancer pagurus has been localized in situ by DNA-DNA hybridization in the nuclei of various spermatogenetic, midgut gland, intestinal and tegument cells. The specificity of hybridization was checked by various tests before, during and after hybridization. The nuclear sites revealed by this method were compared with those shown by quinacrine mustard or Giemsa staining. The A---T-rich satellite DNA appears to be highly dispersed and does not seem to have any preferential localization inside the crab interphasic nucleus. This situation was compared with that presented by mouse nuclei using similar methods.     

Deoxyribonucleic acid of Cancer pagurus. IV. Elution behaviour on hydroxyapatite chromatographic column.

Fractionation of native DNA on hydroxyapatite columns depends, when flat and continuous gradients are used, on the base composition, GC-rich fractions being eluted in the first fractions. Crab satellite DNA behaves abnormally : the first eluted fractions are enriched in poly d(A-T).d(A-T) instead of GC as usual. It amy be suggested that these differences in the behaviour could be attributed to the fact that the secondary structure of crab DNA satellite is different from the secondary structure of the main DNA component.     

Deoxyribonucleic acid of Cancer pagurus. II. Template activity for a DNA-dependent DNA polymerase of eukaryotic cells

The template activity of Cancer pagurus DNA and its two components (poly d(A-T) and main component) in response to a DNA polymerase purified from regenerating rat liver has been studied and compared to the results previously obtained with synthetic templates. In the double-stranded native state, whole crab DNA and the main component were poor templates. Their replication was increased by thermal denaturation and inhibited by actinomycin. Like the synthetic copolymer poly[d(A-T)·d(T-A)], native crab poly d(A-T) could be copied and its duplication was not inhibited by actinomycin. The structural difference between native poly d(A-T) Form I, isolated on a density gradient, and partially renatured poly d(A-T) Form II, isolated on hydroxylapatite, resulted in a modification of their template activity. The kinetic studies of [³H] dGMP and [³H] dAMP incorporation confirmed the importance of single-stranded regions (particulary dC regions) in the initiation of the in vitro duplication.     

The isolation of a plasma membrane-rich fraction from the skeletal muscle of two species of marine crab,Carcinus maenas and Cancer pagurus

• 1.1. A rapid method for the isolation of a plasma membrane-rich fraction from crab leg muscle, with high purity and yield recovery was developed.
• 2.2. The method is based on sodium iodide extraction of the crude homogenate, followed by centrifugation on Percoll self-creating gradient.
• 3.3. (Na⁺K⁺)ATPase and alkaline phosphodiesterase I were used as marker enzymes for the plasma membrane and revealed levels of purification of approximately 13-fold and yields recovery of the total activity in the crude muscle homogenate of approximately 18%, for both species of crab studied.

     

Autoproteolytic stability of a trypsin from the marine crab Cancer pagurus

Autoproteolytic stability is a crucial factor for the application of proteases in biotechnology. In contrast to vertebrate enzymes, trypsins from shrimp and crayfish are known to be resistant against autolysis. We show by characterisation of a novel trypsin from the gastric fluid of the marine crab Cancer pagurus that this property might be assigned to the entire class of crustaceans. The isolated and cloned crab trypsin (C.p.TryIII) exhibits all characteristic properties of crustacean trypsins. However, its overall sequence identity to other trypsins of this systematic class is comparatively low. The high resistance against autoproteolysis was determined by mass spectrometry, which revealed a low susceptibility of the N-terminal domain towards autolysis. By homology modelling of the tertiary structure, the elevated stability was attributed to the distinctly different pattern of autolytic cleavage sites, which is conserved in all known crustacean trypsin sequences.     

The specificity of a neutral deoxyribonuclease from Cancer pagurus.

The recently isolated neutral deoxyribonuclease from crab (Cancer pagurus) testes has been characterized in its mode of action and its specificity. The enzyme is a typical endonuclease, forming 5'-phosphate oligonucleotides of large average size; after extensive digestion of calf thymus DNA over 75% of the fragments have a size larger than pentanucleotides and mononucleotides are absent. As far as specificity is concerned, thymidine is very abundant in the 5'-penultimate position (approximately 50%) and in the 3'-terminal position (37%) and almost absent in the 5'-terminal position (approximately 1%), the values quoted concerning Escherichia coli digests of average size (Pn) between 50 and 10.     

An approach for quantitative assessment of fluorescence in situ hybridization (FISH) signals for applied human molecular cytogenetics.

A number of applied molecular cytogenetic studies require the quantitative assessment of fluorescence in situ hybridization (FISH) signals (for example, interphase FISH analysis of aneuploidy by chromosome enumeration DNA probes; analysis of somatic pairing of homologous chromosomes in interphase nuclei; identification of chromosomal heteromorphism after FISH with satellite DNA probes for differentiation of parental origin of homologous chromosome, etc.). We have performed a pilot study to develop a simple technique for quantitative assessment of FISH signals by means of the digital capturing of microscopic images and the intensity measuring of hybridization signals using Scion Image software, commonly used for quantification of electrophoresis gels. We have tested this approach by quantitative analysis of FISH signals after application of chromosome-specific DNA probes for aneuploidy scoring in interphase nuclei in cells of different human tissues. This approach allowed us to exclude or confirm a low-level mosaic form of aneuploidy by quantification of FISH signals (for example, discrimination of pseudo-monosomy and artifact signals due to over-position of hybridization signals). Quantification of FISH signals was also used for analysis of somatic pairing of homologous chromosomes in nuclei of postmortem brain tissues after FISH with "classical" satellite DNA probes for chromosomes 1, 9, and 16. This approach has shown a relatively high efficiency for the quantitative registration of chromosomal heteromorphism due to variations of centromeric alphoid DNA in homologous parental chromosomes. We propose this approach to be efficient and to be considered as a useful tool in addition to visual FISH signal analysis for applied molecular cytogenetic studies.     

Methylation of satellite sequences in mouse spermatogenic and somatic DNAs.

The distribution of 5-methyl cytosine (5-MeC) residues in a highly repetitive sequence, mouse major satellite, was examined in germinal versus somatic DNAs by digestion with the methylation sensitive isoschizomers Msp I and Hpa II and Southern blot analysis, using a cloned satellite probe. DNA from liver, brain, and a mouse fibroblast cell line, C3H 10T1/2, yielded a multimeric hybridization pattern after digestion with Msp I (and control Eco RI) but were resistant to digestion with Hpa II, reflecting a high level of methylation of the satellite sequences. In contrast, DNA from mature sperm was undermethylated at these same sequences as indicated by the ability of Hpa II to generate a multimeric pattern. DNAs from purified populations of testis cells in different stages of spermatogenesis were examined to determine when during germ cell differentiation the undermethylation was established. As early as in primitive type A, type A, and type B spermatogonia, an undermethylation of satellite sequences was observed. This suggest that this highly specific undermethylation of germ cell satellite DNA occurs very early in the germ cell lineage, prior to entry into meiosis.     

Localization of the genes for silk fibroin in silk gland cells of Bombyx mori.

Radioactive iodinated silk fibroin messenger RNA and ribosomal RNA have been used as probes to localize their genes in tissue sections of Bombyx mori by in situ hybridization. From filter hybridization experiments it is inferred that the majority of the grains produced by in situ hybridization with fibroin mRNA represents specific hybridization to fibroin genes. Sections of the posterior silk gland where silk is synthesized have been compared with those of the middle gland which does not synthesize fibroin. Glands have been analyzed from the second through the fifth (last) larval instar during feeding and moulting periods. During later stages when the gland cells increase their DNA content by polyploidization, serial sections were required to follow the distribution of grains through entire nuclei. At all stages, both ribosomal DNA and fibroin genes are distributed randomly throughout the nuclei without a preferred relationship to any nuclear structure.     

Efficient identification of proteins from ovaries and hepatopancreas of the unsequenced edible crab,Cancer pagurus,by mass spectrometry and homology-based,cross-species searching

Proteome maps of hepatopancreas (midgut gland) and ovarian tissues of the crustacean, Cancer pagurus (Decapoda; edible crab) have been produced by 2D-PAGE and identification of proteins, following trypsin proteolysis, by electrospray MS/MS and database searching. Owing to the lack of sequence information on proteins and fully sequenced genomes amongst the decapod crustaceans and given the evolutionary distance to the nearest full genome database (Daphnia), it was necessary to adopt a non-conventional identification approach. Thus, a strategy was developed for effective identification of decapod proteins by sequence similarity, homology-based cross-species database searching, using various algorithms and a combination of NCBI Crustacea and Arthropoda databases, together with the Arthropoda PartiGene database (Blaxter, University of Edinburgh). In both hepatopancreas and ovary tissues, the largest group of proteins identified were a variety of enzymes, followed by a smaller number of storage/transport proteins [including vitellogenin (yolk protein), several subunits of hemocyanin, cryptocyanin, ferritin and calreticulin], with fewer structural proteins (actin, tubulin) and heat-shock proteins, in addition to a number of proteins of miscellaneous functions. Such protein identifications allow the development of tools, such as antibodies and RNA/DNA probes, to investigate the functions of the proteins in specific tissues during development.     

Interspersion of mouse satellite deoxyribonucleic acid sequences

DNA sequences with homology to the major (A + T)-rich mouse satellite component were localized in CsCl gradients by hybridization with a labeled satellite cRNA probe. Although, as expected, most of the hybridization was to DNA in the satellite-rich shoulder, substantial radioactive cRNA hybridized with DNA from denser regions of the gradient. Further examination revealed that hybridization to main-band DNA was not due to physical trapping of satellite DNA in the gradient, and melting experiments argue that the associated radioactivity was due to true RNA/DNA hybridization. Nearest-neighbor analysis of hybridized [alpha-32P]CTP-labeled l-strand cRNA indicates that hybridization to main-band DNA is by the satellite cRNA and not a contaminant. Together, these data argue that mouse satellite-like sequences are interspersed within the main-band fraction of DNA. For the support of this contention, total mouse DNA, purified main-band DNA, and purified satellite DNA were digested with EcoRI, sedimented in a sucrose gradient, and hybridized with labeled satellite cRNA. Mouse satellite DNA is not cleaved with EcoRI, so that purified EcoRI-digested satellite DNA sediments as a high molecular weight component. When total mouse DNA is digested with EcoRI, the majority of satellite-like sequences remain as high molecular weight DNA; however, significant amounts of satellite-like sequences sediment with the bulk of the lower molecular weight digested DNA, lending further credence to the argument that satellite-like sequences are interspersed with main-band DNA.     

Cytological localization of fast renaturing and satellite DNA sequences inVicia faba

Summary DNA sequences reassociating within a Cot value of 1.8×10^–1 and those producing a light satellite in a CsCl density gradient were isolated fromVicia faba DNA and hybridizedin situ on squashes of roots of the same species. Silver grains were seen to be scattered over both the interphase nuclei and the metaphase chromosomes after hybridization with fast renaturing DNA sequences, indicating these are fairly regularly interspersed in theV. faba genome. Clustered labeling occurred after hybridization with satellite DNA sequences, indicating these are clustered in the genome. The localization of satellite DNA in chromosomes appeared to correspond closely to the position of the bright bands detectable after staining with quinacrine mustard. After hybridization with both DNA probes, labeling intensity over the nuclei of meristematic cells was higher than that over the nuclei of differentiating and/or differentiated cells. These results are discussed in relation to the structure of the cell nucleus, the mechanism of quinacrine banding and to previous data suggesting underrepresentation of nuclear repeated DNA sequences in differentiatingV. faba root cells.     

Enterospora canceri n. gen., n. sp., intranuclear within the hepatopancreatocytes of the European edible crab Cancer pagurus

Only 1 genus (Nucleospora) within 1 family (Enterocytozoonidae) of the Microsporidia contains species that are parasitic within the nuclei of their host cells; to date, all described intranuclear Nucleospora spp. parasitise fish. This study describes the first intranuclear microsporidian parasite of an invertebrate, the European edible crab Cancer pagurus L. (Decapoda: Cancridae). Infected crabs displayed no obvious external signs, and maximum apparent prevalence of infection within a monthly sample was 3.45%. Infected hepatopancreatic tubules were characterised by varying numbers of hypertrophic and eosinophilic nuclei within epithelial cells. Parasite stages appeared as eosinophilic granular accumulations causing margination of host chromatin. In advanced cases, the tubule epithelia degenerated, with parasites and sloughed epithelial cells appearing in tubule lumens. All life stages of the parasite were observed within host nuclei. Uninucleate meronts were not detected, although binucleate stages were observed. Multinucleate plasmodia (sporogonal plasmodia) contained up to 22 nuclei in section, and late-stage plasmodia contained multiple copies of apparatus resembling the polar filament and anchoring disk, apparently associated with individual plasmodial nuclei. As such, aggregation and early assembly of sporoblast components took place within the intact sporogonial plasmodium, a feature unique to the Enterocytozoonidae. Liberation of sporoblasts from plasmodia or the presence of liberated sporoblasts was not observed in this study. However, large numbers of maturing and mature spores (measuring 1.3 +/- 0.02 x 0.7 +/- 0.01 microm) were frequently observed in direct contact with the host nucleoplasm. Considering the shared features of this parasite with microsporidians of the family Enterocytozoonidae, and the unique presence of this parasite within the nucleoplasm of decapod crustacean hepatopancreatocytes, this parasite (Enterospora canceri) is proposed as the type species of a new genus (Enterospora) of microsporidian. Molecular taxonomic work is now required, comparing Enterospora to Enterocytozoon and Nucleospora, the 2 other genera within the Enterocytozoonidae.     

Extranuclear DNA of a Marine Chromophytic Alga : RESTRICTION ENDONUCLEASE ANALYSIS

Two extranuclear DNA species have been isolated from the marine alga Olisthodiscus luteus. Rapid lysis of cells followed by the immediate addition of CsCl to the lysate was critical to the preservation of these satellite DNA species. Restriction endonuclease analysis demonstrates a molecular weight of 99 × 10⁶ for chloroplast DNA and 23 × 10⁶ for a second satellite species. The origin of the second satellite is not known. However, this smaller satellite DNA which originates from a nonnuclear, DNAse insensitive cellular component, displays no sequence homology with ctDNA by hybridization experiments. Constancy of restriction endonuclease fragment patterns of chloroplast and second satellite species during all phases of the growth cycle, whether cultures were maintained synchronously or asynchronously, was demonstrated.     

Sequence-specific microscopic visualization of DNA methylation status at satellite repeats in individual cell nuclei and chromosomes

Methylation-specific fluorescence in situ hybridization (MeFISH) was developed for microscopic visualization of DNA methylation status at specific repeat sequences in individual cells. MeFISH is based on the differential reactivity of 5-methylcytosine and cytosine in target DNA for interstrand complex formation with osmium and bipyridine-containing nucleic acids (ICON). Cell nuclei and chromosomes hybridized with fluorescence-labeled ICON probes for mouse major and minor satellite repeats were treated with osmium for crosslinking. After denaturation, fluorescent signals were retained specifically at satellite repeats in wild-type, but not in DNA methyltransferase triple-knockout (negative control) mouse embryonic stem cells. Moreover, using MeFISH, we successfully detected hypomethylated satellite repeats in cells from patients with immunodeficiency, centromeric instability and facial anomalies syndrome and 5-hydroxymethylated satellite repeats in male germ cells, the latter of which had been considered to be unmethylated based on anti-5-methylcytosine antibody staining. MeFISH will be suitable for a wide range of applications in epigenetics research and medical diagnosis.     

Polymorphic nuclear microsatellite loci for studies of brown crab, Cancer pagurus L

Twelve polymorphic microsatellite DNA loci were isolated from the brown crab, Cancer pagurus L., by construction of microsatellite-enriched genomic libraries. Genotyping of 40 individuals from Norfolk (UK) revealed variable levels of locus polymorphism with an average of 7.75 alleles per locus (range 2-22). The observed and expected heterozygosities per locus ranged from 0.025 to 0.868 and from 0.025 to 0.947, respectively. No evidence of linkage disequilibrium was detected between pairs of loci and genotype proportions at all loci conformed to Hardy-Weinberg equilibrium expectations. The microsatellite loci developed constitute a suite of genetic markers applicable to numerous areas of C. pagurus research.     

The interphase distribution of satellite DNA-containing heterochromatin in mouse nuclei

The distribution of sites capable of binding mouse satellite-complementary RNA in the cytological hybridization reaction has been examined in mouse liver and testis interphase nuclei. The approach taken has been to combine hybridization with semi-thin sectioning and autoradiography in order to obtain a clear picture of the relationship of satellite DNA-containing structures to the rest of the interphase nucleus. In liver nuclei, hybridization occurs primarily with blocks of heterochromatin associated with the nuclear envelope. The most prominent of these, in terms both of size and intensity of hybridization, is the nucleolar stalk and the rest of the nucleolus-associated heterochromatin. The nucleolar body itself is not labeled, nor is much of the peripheral condensed chromatin ; in fact, a polarized distribution of satellite DNA is evident. In Sertoli and spematid nuclei, satellite DNA is found in a small number of large heterochromatin blocks with which the nucleolus is associated; some of this material bears a relationship to the nuclear envelope in these cells also.     

The chromosomal distribution of satellite DNA in the germ-line and somatic tissues of the gall midge,Heteropeza pygmaea

Somatic DNA from Heteropeza pygmaea separated in CsCl gradients into a main band DNA (?=1.685 g/cm³) and a satellite band (?=1.716 g/cm³) comprising 15% of the total DNA. The satellite melted sharply at 93.0°C in SSC, 10.4°C higher than the main band DNA. Satellite DNA reassociated rapidly, banding in CsCl heavier than native satellite but lighter than denatured satellite. The complementary strands of the satellite formed a single band in alkaline gradients and hence are apparently similar in G+T composition. — Filter hybridization experiments with Xenopus ribosomal RNA showed that the satellite band does not contain ribosomal cistrons. — Complementary RNA (cRNA) transcribed in vitro from isolated satellite bound extensively to satellite DNA but not to main band DNA. — The strain of Heteropeza used here contained about 58 chromosomes in germ-line cells and reproduced only paedogenetically. During early cleavage, the presumptive somatic nuclei eliminate most of their chromosomes (E-chromosomes) and retain only ten (S-chromosomes). In situ hybridizations with satellite-cRNA showed satellite DNA (prepared from predominately somatic tissues) to be localized in the centromeric heterochromatin of S- and E-chromosomes. Silver grain comparisons suggested that the amount of satellite is equivalent in both types of chromosomes. — Lastly we found that both diploid and polyploid cells contain similar amounts of satellite DNA. We interpreted this to mean that during polyploidization, as has been demonstrated during polytenization, satellite DNA does not replicate or replicates only slightly while other DNA fractions increase.