A new family of satellite DNA sequences as a major component of centromeric heterochromatin in owls (Strigiformes)

We isolated a new family of satellite DNA sequences from Hae III- and Eco RI-digested genomic DNA of the Blakistons fish owl ( Ketupa blakistoni). The repetitive sequences were organized in tandem arrays of the 174 bp element, and localized to the centromeric regions of all macrochromosomes, including the Z and W chromosomes, and microchromosomes. This hybridization pattern was consistent with the distribution of C-band-positive centromeric heterochromatin, and the satellite DNA sequences occupied 10% of the total genome as a major component of centromeric heterochromatin. The sequences were homogenized between macro- and microchromosomes in this species, and therefore intraspecific divergence of the nucleotide sequences was low. The 174 bp element cross-hybridized to the genomic DNA of six other Strigidae species, but not to that of the Tytonidae, suggesting that the satellite DNA sequences are conserved in the same family but fairly divergent between the different families in the Strigiformes. Secondly, the centromeric satellite DNAs were cloned from eight Strigidae species, and the nucleotide sequences of 41 monomer fragments were compared within and between species. Molecular phylogenetic relationships of the nucleotide sequences were highly correlated with both the taxonomy based on morphological traits and the phylogenetic tree constructed by DNA-DNA hybridization. These results suggest that the satellite DNA sequence has evolved by concerted evolution in the Strigidae and that it is a good taxonomic and phylogenetic marker to examine genetic diversity between Strigiformes species.An erratum to this article can be found at Communicated by Y. Hiraoka     

Two highly repetitive DNA satellites of Drosophila hydei localized within the α-heterochromatin of specific chromosomes

Two major highly repetitive satellites have been isolated from nuclear DNA of Drosophila hydei by sequential centrifugations in Ag⁺/Cs₂SO₄, actinomycin D/CsCl and CsCl. Their CsCl-densities are 1.696 and 1.714 g/cm³. In diploid larval brains they comprise about 13% and 4% respectively of the DNA. Both satellites are localized and chromosome specific. The 1.696 component was shown to be derived from the X-heterochromatin by comparison of different stocks containing different amounts of X-heterochromatin and by in situ hybridization of the ¹²⁵I-labelled light single strand of this satellite. Since the amount of X-heterochromatin equals the amount of this satellite it was concluded that the 1.696 satellite is the only major DNA component of the X-heterochromatin besides minor DNA fractions (e.g. rDNA). The other highly repetitive satellite (1.714 g/cm³) hybridized in situ to all four acrocentric autosome pairs of D. hydei, but neither to the X nor to the small dot-like sixth chromosome, and not to the Y.     

The chromosomal localisation of satellite DNA in Ptyas mucosus (Ophidia,Colubridae)

Ptyas mucosus male DNA has a repetitious DNA satellite (p= 1.700 g cm^?3) constituting 5% of the haploid genome. In situ hybridisation of radioactive complementary RNA (cRNA) has revealed that satellite sequences are located in the centromeric region of one pair of macrochromosomes and in the terminal region of 8 pairs of microchromosomes. These regions are constitutively heterochromatic as revealed by C-banding. The possibility of involvement of satellite rich microchromosomes in nucleolus organisation is discussed.     

Localisation of satellite DNA in the microchromosomes of the Japanese quail by in situ hybridization

In situ hybridisation of radioactive complementary RNA has been used to localise the G-C rich repetitious DNA satellite in chromosomes of the Japanese quail. The satellite sequences are located predominantly in the microchromosomes. No cross hybridisation is found with duck or chicken microchromosomes. The relationship between repetitious DNA, heterochromatin and nucleolus-organisation is discussed.     

Phosphorylation and Localization of Replication Protein A during Oogenesis and Early Embryogenesis of Drosophila melanogaster

The phosphorylation and localization of Drosophila melanogaster Replication Protein A (DRP-A) was examined during oogenesis and in single embryos during the syncytial nuclear divisions of embryogenesis. DRP-A from ovaries was separated by two-dimensional electrophoresis into multiple phosphorylated species that include a previously unresolved form of RP-A. These forms are developmentally regulated with a major phosphorylated form appearing at stage 11 of oogenesis and persisting into mature eggs. Actively cycling early embryos were examined to investigate DNA replication in the absence of repair synthesis due to perturbation by drugs or mutation. An oscillation of the two major forms of DRP-A was observed over multiple cell cycles. The phosphorylated form was most abundant at mitosis and the nonphosphorylated form at interphase. In contrast to other systems where a phosphorylated form of RPA has been correlated with S phase, only the nonphosphorylated form of Drosophila RP-A is observed in early Drosophila embryos during DNA replication. Consistent with this role in DNA metabolism, DRP-A was localized to the nucleus. Subsequently at mitosis, DRP-A becomes delocalized. Strikingly, in ovaries a relatively large amount of DRP-A was observed during the early mitotic stages of oogenesis.     

Characterization and chromosomal distribution of a novel satellite DNA sequence of Japanese quail (Coturnix coturnix japonica)

A novel satellite DNA sequence of Japanese quail (Coturnix coturnix japonica) was isolated from genomic DNA digested with restriction endonuclease, Bg/II. Sequence analysis of three different-size clones revealed the presence of a tandem array of a GC-rich 41 bp repeated element. This sequence was localized by fluorescence in situ hybridization (FISH) primarily to microchromosomes of Japanese quail (2n = 78); approximately 50 of the 66 microchromosomes showed positive signals, although hybridization signals were also detected on chromosomes 4 and W. This satellite DNA did not cross-hybridize with genomic DNA of chicken (Gallus gallus) and Chinese painted quail (Excalfactoria chinensis) under moderately stringent conditions, suggesting that this class of repetitive DNA sequences was species specific and fairly divergent in Galliformes species.     

Nuclear proteins : IV. Deficiency of non-histone proteins in condensed chromatin of Drosophila virilis and mouse

Drosophila virilis egg nuclei were fractionated by a technique of multiple sonication and centrifugation in an isotonic buffer containing 0.15 M NaCl, Mg²⁺ and Ca²⁺. This allowed the condensed chromatin to remain tightly condensed. By Hoechst 33258 staining this procedure resulted in brightly fluorescing and poorly fluorescing fractions. The brightly fluorescing fraction was enriched in satellite DNA. Examination of the non-histone proteins by SDS slab gel electrophoresis showed that this fraction was markedly deficient in non-histone proteins and contained no unique major non-histone proteins. The poorly fluorescing fractions were enriched in non-histone proteins. Similar results were obtained with mouse liver nuclei. Comparison of the non-histone proteins of D. virilis (40% satellite DNA), D. americana (35% satellite DNA), and D. ezoana (no satellites) confirmed the absence of major, satellite specific, non-histone proteins. These results, suggesting condensed chromatin is primarily a DNA-histone complex, agree with published cytochemical studies.     

Synthesis and characterization of amplified DNA in oocytes of the house cricket,Acheta domesticus (Orthoptera: Gryllidae)

At a time in the life cycle when a large proportion of the oocytes of Acheta incorporate ³H-thymidine into an extrachromosomal DNA body, synthesis of a satellite or minor band DNA, the density of which is greater than main band DNA, is readily detected. Synthesis of the satellite DNA is not detectable in tissues, the cells of which do not have a DNA body, or in ovaries in which synthesis of extrachromosomal DNA by the oocytes is completed. The DNA body contains the amplified genes which code for ribosomal RNA. However, less than 1 percent of the satellite DNA, all of which appears to be amplified in the oocyte, is complementary to ribosomal 18S and 28S RNA. In situ hybridization demonstrates that non-ribosomal elements, like the ribosomal elements of the satellite DNA, are localized in the DNA body.Abbreviations used rRNA ribosomal RNA, includes 18S and 28S RNA - rDNA gene sequences complementary to rRNA - cRNA complementary RNA synthesized in vitro     

Studies of microchromosomes and a G-C rich DNA satellite in the quail

The microchromosomes of Japanese quail fibroblasts are shown to be heterochromatic and nucleolus-organizing. Autoradiographic studies indicate that although some DNA replication takes place early, the time of intense replication is in the late S period after most replication in the macrochromosomes has ceased. Analytical centrifugation of quail DNA demonstrated a main band with a buoyant density of 1.701 g/cm³ and a satellite constituting about 5% of the DNA with a buoyant density of 1.715. The G-C content of the main and satellite band was 42 and 55 percent respecively by both buoyant density and DNA Tm. The satellite band renatured much more rapidly than main band DNA indicating it was composed of highly repetitive sequences. When purified satellite DNA was centrifuged at pH 13 it separated into three portions, a major central band constituting 72% of the satellite DNA, and two smaller bands, one heavier and one lighter than the central band, each constituting 14% of the satellite DNA. This indicated that in a portion of the satellite DNA the bases were non-randomly distributed in the half-DNA helices.Supported by N.I.H. Grant GM-15886, The Charles and Henrietta Detoy Research Fellowship.     

Two AT-rich satellite DNAs in the chironomid Glyptotendipes barbipes (Staeger)

Two AT-rich satellite DNAs are present in the genome of Glyptotendipes barbipes. The two satellites have densities of 1.680 g/cm³ (=21% GC) and of 1.673 g/cm³ (=13% GC) in neutral CsCl-density gradients. The main band DNA has a density of 1.691 g/cm³ (=32% GC). This value is in agreement with the 33% GC-content of G. barbipes DNA calculated from thermal denaturation (T_M=83° C). — In brain DNA as well as in salivary gland DNA the two satellite sequences together comprise 12–15% of the total G. barbipes DNA. Comparisons of the density profiles of DNA extracted from polytene and non-polytene larval tissue gave no hints for underreplication of the satellite DNAs during polytenization. — The two satellite DNAs have been isolated from total DNA by Hoechst 33258-CsCl density centrifugation and then localized in the polytene salivary gland chromosomes by in situ hybridization. Both satellite sequences hybridize to all heterochromatic centromere bands of all four chromosomes of G. barbipes. Satellite I (1.673 g/cm³) hybridizes mainly with the middle of the heterochromatin, satellite II (1.680 g/cm³) hybridizes with two bands at the margin of the heterochromatin. In situ hybridization with polytene chromosomes of Chironomus thummi revealed the presence of G. barbipes satellite sequences also in the Ch. thummi genome at various locations, mainly the centromere regions.     

Observations on centromeric heterochromatin and satellite DNA in salamanders of the genus Plethodon

DNA from Plethodon cinereus cinereus separates into two fractions on centrifugation to equilibrium in neutral CsCl. The smaller of these fractions has been described as a high-density satellite. It represents about 2% of nuclear DNA from this species, and it has a density of 1.728 g/cm³. It is cytologically localized near the centromeres of all 14 chromosomes of the haploid set. In P. c. cinereus the heavy satellite DNA constitutes about 1/4 of the DNA in centromeric heterochromatin. The nature of the rest of the DNA in centromeric heterochromatin is unknown. The number of heavy satellite sequences clustered around the centromeres in a chromosome from P. c. cinereus is roughly proportional to the size of the chromosome, as determined by in situ hybridization with satellite-complementary RNA, and autoradiography. Likewise the amount of contromeric heterochromatin, as identified by its differential stainability with Giemsa, shows a clear relationship to chromosome size. — The heavy satellite sequences identified in DNA from P. c. cinereus are also present in smaller amounts in other closely related forms of Plethodon. Plethodon cinereus polycentratus and P. richmondi have approximately half as many of these sequences per haploid genome as P. c. cinereus. P. hoffmani and P. nettingi shenandoah have about 1/3 as many of these sequences as P. c. cinereus. P. c. cinereus, P. c. polycentratus, and P. richmondii all have detectable heavy satellites with densities of 1.728 g/cm³. Among these forms, satellite size as determined by optical density measurements, and number of satellite sequences as determined from hybridization studies, vary co-ordinately. P. c. cinereus heavy satellite sequences are not detectable in P. nettingi, P. n. hubrichti, or P. dorsalis. The latter species has a heavy satellite with a density of 1.718 g/cm³, representing about 8% of the genomic DNA, and two light satellites whose properties have not been investigated. The heavy satellite of P. dorsalis is cytologically localized in the centromeric heterochromatin of this species. — These observations are discussed in relation to the function and evolution of highly repetitive DNA sequences in the centromeric heterochromatin of salamanders and other organisms.     

Methylation patterns of repetitive DNA sequences in germ cells of Mus musculus.

The major and the minor satellite sequences of Mus musculus were undermethylated in both sperm and oocyte DNAs relative to the amount of undermethylation observed in adult somatic tissue DNA. This hypomethylation was specific for satellite sequences in sperm DNA. Dispersed repetitive and low copy sequences show a high degree of methylation in sperm DNA; however, a dispersed repetitive sequence was undermethylated in oocyte DNA. This finding suggests a difference in the amount of total genomic DNA methylation between sperm and oocyte DNA. The methylation levels of the minor satellite sequences did not change during spermiogenesis, and were not associated with the onset of meiosis or a specific stage in sperm development.     

Monomers of a Satellite DNA Sequence of Chaffinch (Fringilla coelebs L., Aves: Passeriformes) Contain Short Clusters of the TTAGGG Repeat

A novel repeated sequence of chaffinch (Fringilla coelebs) designated as GS was isolated from genomic DNA after in vitro amplification of satellite DNA sequences using GSP–PCR technique. The proportion of this repeat in the chaffinch genome constitutes about 0.2%. Monomers are 176 to 199 bp in size and contain a short cluster of the TTAGGG telomeric tandem repeat. The oligomer of the telomeric hexanucleotide is flanked by the sequences that are significantly different in different monomers. The GS sequences are organized as tandemly repeated units and located in a number of chromomycin-positive blocks on the long arms of macrochromosomes 1, 2, 3, 5, and 6, as well as on several microchromosomes. The sequences homologous to the GS satellite of chaffinch were not found in the genomes of redwing (Turdus iliacus) and house sparrow (Passer domesticus).     

Nucleolar DNA in sea urchin oogenesis studied by H-actinomycin D binding

³H-actinomycin D binding to nucleolar DNA was studied during eight successive stages of oocyte differentiation in Strongylocentrotus purpuratus. As the nucleolus increases in size, there is a threefold increase in the amount of ³H-actinomycin D binding to nucleolar DNA. This data is consistent with the interpretation that limited nucleolar gene amplification may occur in sea urchin oogenesis.     

Composition and synthesis of DNA in synchronously growing cells of Chlorella pyrenoidosa

Summary The composition and synthesis of DNA in synchronous cultures of Chlorella pyrenoidosa strain 211/8b has been investigated. Analytical CsCl density gradient centrifugation gave a homogenous major DNA component with a (G+C) content of 51% and a minor component containing 28% (G+C). The (G+C) contents derived from melting profiles were 2–3% lower. A second minor component with approximately 41% (G+C) content was inferred from banding patterns of labelled DNA in preparative CsCl density gradients. ¹⁴C-uracil was readily incorporated into the pyrimidine moieties of the major (nuclear) DNA between the 10th and 18th hour after beginning of the light period, but not at any other time. ¹⁴C-uracil incorporation into the minor (satellite) component was low but continuous throughout the whole cell cycle. The incorporation is correlated with an increase in the proportion of satellite DNA from 6% up to 20% during the time when no nuclear DNA replication takes place. The results suggest that different regulatory mechanisms exist for the nuclear and for satellite DNA synthesis.     

Dynamic distribution of region-specific maternal protein during oogenesis and early embryogenesis of Xenopus laevis

Summary For analysing spatial distribution of maternal proteins in an amphibian egg, monoclonal antibodies specific to certain regions were raised. One monoclonal antibody was found (MoAB Xa5B6) which reacted specifically with the animal hemisphere of the mature Xenopus laevis egg. The maternal protein that reacted with the MoAb Xa5B6 was shown to be distributed asymmetrically along the dorso-ventral axis in the upper region of the equatorial zone of the fertilized egg. At late blastula stage, the antigen protein could be observed clearly in both the marginal zone and animal cap. It was localized predominantly in mesodermal and ectodermal cells of late neurula embryos. The Xa5B6 antigen accumulated during oogenesis. The distribution pattern of maternal protein was remarkably different in the developmental stages of the oocyte. The pattern in the mature oocyte was completely different from that of the immature egg in which the antigen was located in the radial striations of the oocyte cytoplasm. After maturation, the distribution pattern changed drastically to an animal-vegetal polarization and the striation labellings were no longer observed. By Western blot examination, it was confirmed that the amounts of antigen protein were constant during early embryogenesis and the mesoectoderm contained a greater amount of antigens than the endoderm at late blastula. The antibody detected two bands of approximately 70 × 10³ and 30 × 10³ Mr by Western blot analysis. The latter molecule may possibly be a degrading moiety of the former. The results were discussed in relation to establishment of animal-vegetal (A/V) and dorso-ventral (D/V) polarization at the molecular level. Offprint requests to: A.S. Suzuki     

The <Emphasis Type="Italic">Drosophila</Emphasis> RNA-binding protein Lark is required for localization of Dmoesin to the oocyte cortex during oogenesis

The RNA-binding protein Lark has an essential maternal role during Drosophila oogenesis. Elimination of maternal expression results in defects in cytoplasmic dumping and actin cytoskeletal organization in nurse cells. The function of this protein is dependent on the activity of one or more N-terminal RNA-binding domains. Here, we report the identification of Dmoesin (Dmoe) as a candidate RNA target of Lark during oogenesis. In addition to actin defects in the nurse cells of lark mutant ovaries, we observed mislocalization of posteriorly localized mRNAs including oskar and germ cell less in the developing oocyte. Anteriorly and dorsally localized mRNAs were not affected. In addition, we observed displacement of the actin cytoskeleton from the oocyte plasma membrane. These phenotypes are reminiscent of mutations in Dmoe and suggested that this RNA maybe a potential target of Lark. We observed a significant decrease in Dmoe protein associated with the membrane of the developing oocyte with no changes in expression or localization within the nurse cells. Evidence for an association between Lark protein and moe RNA during oogenesis comes from results of a microarray-based Ribonomics approach to identify Lark RNA targets. Thus, our results provide evidence that Dmoe RNA is a target of Lark during oogenesis and that it likely regulates either the splicing or translation of this RNA. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A species specific satellite DNA family of Drosophila subsilvestris appearing predominantly in B chromosomes

This paper describes a species specific satellite DNA family (pSsP216) of Drosophila subsilvestris, a palearctic species of the D. obscura group. The pSsP216 family consists of tandemly arranged 216 bp repetitive units that are predominantly localized on B chromosomes. These chromosomes appear in variable numbers in the karyotype of this species. Some pSsP216 repeats can also be detected in the centromeric heterochromatin of the acrocentric A chromosomes. Two strains, one with and the other without B chromosomes, were investigated for sequence variability and for the location of this satellite DNA on the chromosomes. Among 16 clones of the 216 bp basic repeat unit an overall similarity of about 93% and no strain specific differences were found, indicating that the B chromosomes may have derived from the A chromosomes (probably the dots) by spontaneous amplification of the pSsP216 satellite DNA family.     

Construction of a YAC library from a Beta vulgaris fragment addition and isolation of a major satellite DNA cluster linked to the beet cyst nematode resistance locus Hs1 pat−1

A YAC library was constructed from the Beta vulgaris fragment addition AN5-203b. This monosomic fragment addition harbors an approximate 12-Mbp fragment of B.patellaris chromosome 1 accomodating the Hs1 ^pat-1 conferring resistance to the beet cyst nematode (Heterodera schachtii). The YAC library consists of 20,000 YAC clones having an average size of 140 kb. Screening with organelle-specific probes showed that 12% of the clones contain chloroplast DNA while only 0.2% of the clones hybridizes with a mitochondrial specific probe. On the basis of a sugar beet haploid genome size of 750 Mbp this library represents 3.3 haploid genome equivalents. The addition fragment present in AN5-203b harbors a major satellite DNA cluster that is tightly linked to the Hs1 ^pat-1 locus. The cluster is located on a single 250-kb EcoRI restriction fragment and consists of an estimated 700–800 copies of a 159-bp core sequence, most of which are arranged in tandem. Using this core sequence as a probe, we were able to isolate 1 YAC clone from the library that contains the entire 250-kb satellite DNA cluster.Abbreviations YAC Yeast artificial chromosome - BCN beet cyst nematode - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism