Liver chromatin fractions inMus andAkodon

Summary The liver chromatin fromMus musculus andAkodon molinae was separated in 8 fractions by differential centrifugation. Like fractions from both species showed approximately similar contents of DNA, equivalent ratios of histone to non-histone proteins, corresponding template activities and equal amounts of positive C-banded material. On the other hand, heavy chromatin fractions ofMus were highly enriched in satellite DNA whereas no satellite DNA was found inAkodon chromatin. Heavy chromatin fractions isolated by differential sedimentation have been usually homologued with the constitutive heterochromatin. The properties of the constitutive chromatin are discussed and the validity of the foregoing concept is challenged. It is proposed to define the constitutive heterochromatin as those chromatin regions comprising highly repeated DNA sequences clustered in restricted areas of chromosomes and not transcribed (satellite DNA).     

Screening of isolated DNA for sequences released from anchorage sites in nuclear matrix

Isolated chromosomal DNA is associated with polypeptides that are not released from DNA by several methods designed to purify DNA, e.g. treatment with sodium dodecyl sulphate. DNA fragments associated with these very tight DNA/protein complexes show high affinity to nitrocellulose filters in the presence of salt concentrations of 500 mM or greater. Consequently, a fraction of AluI-fragmented native DNA comprising the complexes and 0.2 to 0.3 micron of vicinal DNA can be isolated by one filtration step. This fraction of DNA shows characteristics of residual DNA sequences retained in nuclei after extraction with nucleases and high salt (nuclear matrix). The DNA fragments retained on filters are highly enriched in replicative DNA; and their degree of hybridization with poly(A)+ RNA points to enrichment in actively transcribed sequences. The results support previous work indicating that the very tight DNA/polypeptide complexes co-isolating with DNA under conditions that release other peptide materials from DNA may be anchorage sites of DNA in the nuclear matrix. Moreover, the method described here allows isolation of replicating and actively transcribed DNA sequences directly from isolated total genomic DNA by skipping artefact-prone isolations of the nuclear matrix.     

Cloning and characterization of genomic DNA sequences of four self-incompatibility alleles in sweet cherry (<Emphasis Type="Italic">Prunus avium</Emphasis> L.)

Gametophytic self-incompatibility (GSI) in sweet cherry is determined by a locus S with multiple alleles. In the style, the S-locus codifies for an allele-specific ribonuclease (S-RNase) that is involved in the rejection of pollen that carries the same S allele. In this work we report the cloning and genomic DNA sequence analysis including the 5 flanking regions of four S-RNases of sweet cherry (Prunus avium L., Rosaceae). DNA from the cultivars Ferrovia, Pico Colorado, Taleguera Brillante and Vittoria was amplified through PCR using primers designed in the conserved sequences of sweet cherry S-RNases. Two alleles were amplified for each cultivar and three of them correspond to three new S-alleles named S ₂₃ , S ₂₄ and S ₂₅ present in 'Pico Colorado', 'Vittoria' and 'Taleguera Brillante' respectively. To confirm the identity of the amplified fragments, the genomic DNA of these three putative S-RNases and the allele S ₁₂ amplified in the cultivar Ferrovia were cloned and sequenced. The nucleotide and deduced amino-acid sequences obtained contained the structural features of rosaceous S-RNases. The isolation of the 5-flanking sequences of these four S-RNases revealed a conserved putative TATA box and high similarity among them downstream from that sequence. However, similarity was low compared with the 5-flanking regions of S-RNases from the Maloideae. S ₆ - and S ₂₄ -RNase sequences are highly similar, and most amino-acid substitutions among these two RNases occur outside the rosaceous hypervariable region (RHV), but within another highly variable region. The confirmation of the different specificity of these two S-RNases would help elucidate which regions of the S-RNase sequences play a role in S-pollen specific recognition.Communicated by H.F. Linskens     

A male-specific nuclease-resistant chromatin fraction in the mealybug Planococcus lilacinus

In mealybugs, chromatin condensation is related to both genomic imprinting and sex determination. The paternal chromosomal complement is condensed and genetically inactive in sons but not in daughters. During a study of chromatin organization in Planococcus lilacinus, digestion with micrococcal nuclease showed that 3% to 5% of the male genome is resistant to the enzyme. This Nuclease Resistant Chromatin (NRC) apparently has a nucleosomal organization. Southern hybridization of genomic DNA suggests that NRC sequences are present in both sexes and occur throughout the genome. Cloned NRC DNA is A+T-rich with stretches of adenines similar to those present in mouse -satellite sequences. NRC DNA also contains sequence motifs that are typically associated with the nuclear matrix. Salt-fractionation experiments showed that NRC sequences are matrix associated. These observations are discussed in relation to the unusual cytological features of mealybug chromosomes, including the possible existence of multiple centres of inactivation.     

Genomic organization of the canrep repetitive DNA in Brassica juncea

Canrep is a heterogeneous, tandemly repeated, 176 bp nucleotide sequence that contains a single Hind III site and is present in high copy numbers in the genomes of many Brassica species. Complete clusters of repeats of this DNA were cloned from the nuclear DNA of Brassica juncea. Restriction-fragment dimers and higher multimers of the 176 bp sequence have arisen by mutations within the Hind III recognition sequence. Adjacent repeats from within the same cluster usually have different nucleotide sequences with features indicating that diversity is generated by a mechanism that causes site-specific base substitutions. While most of the units of canrep DNA are clustered in long arrays of tandem repeats, some are dispersed throughout the genome as isolated copies or in small clusters. Regardless of the size of the arrays, each cluster begins and ends with a variable-length, truncated repeat and is flanked by inverted copies of the sequence 5-ATCTCAT3-,which is not part of the basic sequence of the canrep family of DNAs. Furthermore, some clusters are located close to nucleotide sequences related to those of known plant transposons. Thus, canrep elements may be dispersed by transposition. There are two distinct subfamilies of canrep sequences in B. juncea, and one of these is closely related to one of the two subfamilies of this type of DNA from B. napus, indicating that it originated from B. campestris, the common diploid ancestor of both amphidiploid species. Neither the repetitive DNA nor nucleotide sequences flanking canrep clusters are transcribed in seedlings, suggesting that even small arrays of repeats are located in heterochromatic regions and might be involved in chromatin condensation and/or chromosome segregation.     

The properties of oligonucleotide fragments containing the triphosphorylated 5′-termini of nuclear pre-mRNA from Ehrlich ascites carcinoma cells

Triphosphorylated 5-end fragments 50–150 nucleotides in length were isolated from nuclear pre-mRNA with the aid of a hydroxyapatite chromatography. They are enriched in U and G (28 and 26%, respectively). About 15% of the fragments isolated from poly(U)⁺RNA contain poly(U) tracks. Neither poly(A)-nor double-stranded sequences were found. Hybridization experiments in conditions of vast DNA excess demonstrated that the 5-end fragments contain a low amount of highly repetitive sequences but enriched in sequences hybridizing at C ₀ t 1/2 ~100.     

Satellite DNA properties of the germ line limited DNA and the organization of the somatic genomes in the nematodesAscaris suum andParascaris equorum

During the early cleavage divisions in some Ascarids, parts of the chromosomes are eliminated from the somatic blastomeres (chromatin diminution, Boveri, 1887) while the chromosomes in the germ line cells maintain their integrity. To characterize the germ line and soma genome, DNA was isolated from gametes and embryonic somatic cells of two Ascarid species,Parascaris equorum var. univalens andAscaris suum. It was shown that the germ line limited DNAs of these species have the same density and almost identical reassociation kinetics: in CsCl the predominant component of the germ line limited DNA ofP. equorum andA. suum has the buoyant density of 1.697g/cm³, while soma DNA of both species bands at 1.700 g/cm³. InP. equorum there is a small additional germ line limited satellite DNA component with the density of 1.690 g/cm³, identical to that of mitochondrial DNA of both organisms. Comparison of the reassociation kinetics of germ line and soma DNA demonstrates for both species that the eliminated DNA sequences are highly repetitive. In contrast to these similarities between the germ line limited DNAs ofP. equorum andA. suum the analysis of their base composition revealed differences (40% guanine plus cytosine inP. equorum and 36% inA. suum). The only very fast reassociating DNA sequences which we could isolate from soma DNA was demonstrated to be foldback DNA. The reassociation kinetics of totalA. suum soma DNA was investigated by hydroxylapatite chromatography. Least squares analysis of the data revealed about 10% of intermediate repetitive DNA sequences. Their interspersion between single copy DNA sequences was analyzed by comparing the reassociation kinetics of DNA fragments 0.35 and 7.2 kilobases long. Thus the DNA sequence arrangement ofAscaris does not follow the short period interspersion pattern observed in most organism.     

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.     

Quantification and characterization of nuclear genomes in commercial red seaweeds (Gracilariales) from the Philippines

Eight species of Gracilariaceae from the Philippines, representing the generaGracilaria, Gracilariopsis andHydropuntia, were investigated to quantify and characterize their nuclear genomes. DNA reassociation kinetics were used to determine nuclear genome organization and complexity in six of these species. Results indicate the presence of three second order components corresponding to fast, intermediate and slow fractions. Repetitive sequences varied from 13–74% and unique DNA ranged from 26–84%. Microspectrophotometry with the DNA-localizing fluorochrome DAPI was used to quantify nuclear DNA contents. Comparisons of mean nuclear DNA (I _f ) values to chicken erythrocytes (RBC) resulted in an estimate of 0.38–0.43 pg/2 C genomes for seven of the species investigated. Preliminary analyses of agar content and quality confirm the economic potential ofGracilaria firma, Gracilaria sp. 2 from Sorsogon andGracilariopsis bailinae. Nuclear genome profiles developed from data for genome size, organization and complexity are compared with data for agar quantity and quality. Gel quality and quantity do not appear to be correlated with either large repetitive fraction DNA or a high degree of genome complexity.Author for correspondence     

Different modes of state transitions determine pattern in the Phosphatidylinositide-Actin system

Background

In the interphase nucleus of metazoan cells DNA is organized in supercoiled loops anchored to a nuclear matrix (NM). There is varied evidence indicating that DNA replication occurs in replication factories organized upon the NM and that DNA loops may correspond to the actual replicons in vivo. In normal rat liver the hepatocytes are arrested in G0 but they synchronously re-enter the cell cycle after partial-hepatectomy leading to liver regeneration in vivo. We have previously determined in quiescent rat hepatocytes that a 162 kbp genomic region containing members of the albumin gene family is organized into five structural DNA loops.

Results

In the present work we tracked down the movement relative to the NM of DNA sequences located at different points within such five structural DNA loops during the S phase and after the return to cellular quiescence during liver regeneration. Our results indicate that looped DNA moves sequentially towards the NM during replication and then returns to its original position in newly quiescent cells, once the liver regeneration has been achieved.

Conclusions

Looped DNA moves in a sequential fashion, as if reeled in, towards the NM during DNA replication in vivo thus supporting the notion that the DNA template is pulled progressively towards the replication factories on the NM so as to be replicated. These results provide further evidence that the structural DNA loops correspond to the actual replicons in vivo.     

Proteomic analysis of the nuclear matrix in the early stages of rat liver carcinogenesis: Identification of differentially expressed and MAR-binding proteins

Tumor progression is characterized by definite changes in the protein composition of the nuclear matrix (NM). The interactions of chromatin with the NM occur via specific DNA sequences called MARs (matrix attachment regions). In the present study, we applied a proteomic approach along with a Southwestern assay to detect both differentially expressed and MAR-binding NM proteins, in persistent hepatocyte nodules (PHN) in respect with normal hepatocytes (NH). In PHN, the NM undergoes changes both in morphology and in protein composition. We detected over 500 protein spots in each two dimensional map and 44 spots were identified. Twenty-three proteins were differentially expressed; among these, 15 spots were under-expressed and 8 spots were over-expressed in PHN compared to NH. These changes were synchronous with several modifications in both NM morphology and the ability of NM proteins to bind nuclear RNA and/or DNA containing MARs sequences. In PHN, we observed a general decrease in the expression of the basic proteins that bound nuclear RNA and the over-expression of two species of Mw 135 kDa and 81 kDa and pI 6.7-7.0 and 6.2-7.4, respectively, which exclusively bind to MARs. These results suggest that the deregulated expression of these species might be related to large-scale chromatin reorganization observed in the process of carcinogenesis by modulating the interaction between MARs and the scaffold structure.     

The small chromatin fragments released by micrococcal nuclease from hepatoma tissue cultured cell nuclei are strongly enriched in coding DNA sequences and are related to an actively transcribed single-stranded DNA fraction

It was shown with the use of specific probes that mild micrococcal nuclease digestion releases from chromatin actively-transcribed genes as small nucleosome oligomers. In the present work we demonstrate that most if not all of the active genes are accessible to the nuclease. It was found that the short released fragments are greatly enriched in transcribed DNA sequences, the most enriched being the dimers of nucleosomes since 35% of their DNA could be hybridized to cytoplasmic RNA. The results of cDNA-DNA hybridizations indicate that the monomers and dimers of nucleosomes contain most of the DNA sequences which encode poly(A⁺) RNAs, however larger released fragments include some transcribed sequences, while the nuclease-resistant chromatin is considerably impoverished in coding sites. These evidences and the finding that about 25% of the DNA from the dimers of nucleosomes are exclusively located in this class of fragments, tend to prove that the active chromatin regions are attacked in a non-random way by micrococcal nuclease. We have previously isolated, without using exogenous nuclease, an actively transcribed genomic fraction amounting to 1.5–2% of the total nuclear DNA, formed of single-stranded DNA. In the present study we show that all or nearly all the single-stranded DNA sequences could be reassociated with the DNA fragments present in the released monomers and dimers of nucleosomes. Our observations confirmed our previous finding that the greatest part of single-stranded DNA selectively originates from the coding strand of genomic DNA.     

Globin evolution in the genusXenopus: Comparative analysis of cDNAs coding for adult globin polypeptides ofXenopus borealis andXenopus tropicalis

Summary Globin mRNAs ofXenopus borealis andXenopus tropicalis have been cloned and sequenced. The nucleotide and derived amino acid sequences were compared with each other and with already available data fromXenopus laevis. This analysis rendered clear evidence that the common ancestor ofX. laevis andX. borealis, but not ofX. tropicalis, had lost one amino acid of the -globins prior to a genome duplication event that preceded the segregation of the former two species. Replacement-site substitutions were used to calculate a rough time scale of genome duplication and species segregation. The results suggest an ancient separation between theX. laevis and theX. tropicalis groups occurring approximately 110–120 million years ago. Analysis of the amino acid chains demonstrated various alterations. However, some functional domains, like heme-binding sites and₁₂ contact sites, were subject to a high degree of conservation, indicating the existence of functional constraints on them also in the genusXenopus.     

Amplified ribosomal DNA in meiotic prophase oocyte nuclei of acipenserid fishes

Summary In situ hybridization has been performed in sections through ovaries ofAcipenser ruthenus andAcipenser güldenstädti in order to detect the rDNA sequences. Hybridization resulted in specific labelling of the caps of extrachromosomal DNA present in pachytene oocyte nuclei and of the chromatin granules distributed beneath the nuclear envelope in early diplotene nuclei. In the same sections, the nuclei of all ovarian cells in both species (oogonia, leptotene, and zygotene stage oocytes, follicular cells, connective tissue cells) showed a very low, but similar labelling.Amplification of genes for rRNA thus occurs at the pachytene stage in early oogenesis ofAcipenseridae. No rDNA amplification could be detected in the previous stages.     

Satellite DNA specific to knob heterochromatin inCucumis metuliferus (Cucurbitaceae)

Buoyant density gradient analysis of nuclear DNA of fourCucumis species showed asymmetric profiles indicating the presence of satellite DNA sequences in the nuclear genome. A highly repeated satellite DNA sequence was isolated from the nuclear genome ofC. metuliferus under neutral CsCl gradients. The satellite DNA constitutes about 4.96% of total nuclear DNA and has 48.06% guanine plus cytosine content. The kinetic complexity of satellite DNA is 150 times smaller than T₄ phage DNA and the base sequence divergence is low.³H-labeled cRNA transcribed from satellite DNA hybridized clearly to six heterochromatic knobs of pachytene chromosomes. The knob heterochromatin can be distinguished by Giemsa C-banding of pachytene chromosomes. Restriction enzyme analysis and Southern blot hybridization indicated that the satellite DNA has a tandem arrangement and predominantly formed two bands of size 210 and 151 base pairs. Absence of knob satellite DNA ofC. metuliferus in the nuclear genomes ofC. melo, C. anguria andC. sativus showed thatC. metuliferus remains isolated within the genusCucumis.     

Investigation of histone proteins in plant nuclei possessing different ultrastructural organization

Summary According to Nagl and Fusenig (1979) the structure and ultrastructure of plant nuclei is species-specific and is determined by the DNA (2C) value and the amount of the repetitive DNA. Light and electron microscopic observations ofZea mays L.,Pisum sativum L., andPhaseolus vulgaris L. nuclei led us to define their organization as chromonematic, chronomeric and chromocentric, respectively. Nuclear proteins, soluble in 0.4N H₂SO₄ and 0.74M HC1O₄, were extracted from isolated nuclei and resolved according to their solubility and mobility in SDS and acetic acid-urea PAGE and 2D-Triton X 100 PAGE. Differences in the variants (and modifications) of the H 1 histone class and the nucleosomal H 2 A, H 2 B, and H 3 isoforms probably reflect that species-specific nuclear ultrastructure is based, not only on the heterogeneity and the quantity of DNA, but also on the diversity of the protein component of chromatin.Abbreviations MES Morpholinoethane sulfonic acid - PMSF phenylmethylsulphonyl fluoride - DMSO dimethylsulfoxid - SDS sodium dodecylsulfate - TEMED N, N, N N-tetramethylethylen-diamin - PAGE polyacrylamide gel electrophoresis     

Influence of Genotype on Endoreduplication of Giant Chromosomes and Expressivity of Character eyelessin Drosophila melanogaster

In Drosophila melanogaster lines LA (low activity), HA (high activity), andOregon-R (wild type), the effect of genetic background on endoreplication in giant chromosomes of salivary glands, fecundity, and expression of mutation eywas studied. The degree of chromosome polyteny and the number of adult flies in saturated lines ey _L, ey _H, and ey _Or were significantly higher than in the original lines. The degree of chromosome polyteny was correlated with fecundity. The expressivity of the ey character was shown to be far lower in lines ey _L, ey _H, and ey _Or than in the original eyelessline. In the saturated lines, the eye facets were reduced to a similar degree. All the lines studied displayed clear-cut sexual distinctions in this parameter. In the ey _L line, the coefficients of variation for the degree of chromosome polyteny, fecundity, and expressivity of the ey mutation were much lower than in lines eyeless, ey _LAandey _Or.