Heterochromatin and highly repeated DNA sequences in rye (Secale cereale)

Secale cereale DNA, of mean fragment length 500 bp, was fractionated by hydroxylapatite chromatography to allow recovery of a very rapidly renaturing fraction (C₀t 0–0.02). This DNA fraction was shown to contain several families of highly repeated sequence DNA. Two highly repeated families were purified; (1) a fraction which renatured to a density of 1.701 g/ cc and comprised 2–4% of the total genome, and (2) polypyrimidine tract DNA which comprised 0.1% of the total genome. The 1.701 g/cc DNA consisted of short sequence repeat units (5–50 bp long) tandemly repeated in blocks 30 kb long, while a portion of the polypyrimidine tract DNA behaved as part of a much larger block of tandemly repeated sequences. The chromosomal location of these sequences was determined by the in situ hybridisation of radioactive, complementary RNA to root tip mitotic chromosomes and showed the 1.701 g/cc sequences to be largely limited to the telomeric blocks of heterochromatin, accounting for 25–50% of the DNA present in these parts of the chromosomes. The polypyrimidine tracts were distributed at interstitial locations with 20–30% of the sequences at three well defined sites. The combined distributions of the 1.701 g/cc DNA sequences and polypyrimidine tracts effectively individualised each rye chromosome thus providing a sensitive means of identifying these chromosomes. The B chromosomes present in Secale cereale cv. Unevita, did not show defined locations for the sequences analysed. — The data are discussed in terms of the structure of the rye genome and the generality of the observed genomic arrangement of highly repeated sequence DNA.     

Interrelationship of cultivated rices Oryza sativa and O. glaberrima with wild O. perennis complex

Summary Phylogenetic relationship of the cultivated rices Oryza sativa and O. glaberrima with the O. perennis complex, distributed on the three continents of Asia, Africa and America, and O. australiensis has been studied using Fraction 1 protein and two repeated DNA sequences as markers. Fraction 1 protein isolated from the leaf tissue of accessions of different species was subjected to isoelectric focusing. All the species studied have similar nuclear-encoded small subunit polypeptides and chloroplast-encoded large subunit polypeptides, except two of the O. perennis accessions from South America and O. australiensis, which have a different pattern for the chloroplast subunit. Two DNA sequences were isolated from Eco R1 restriction endonuclease digests of total DNA from O. sativa. One of the sequences has been characterized as highly repeated satellite DNA, and the other one as a moderately repeated DNA sequence. These sequences were used as probes in DNA/DNA hybridization with restriction endonuclease digested DNA from some accessions of the different species. Those accessions that are divergent for large subunit polypeptides of Fraction 1 protein (O. australiensis and two of the four South American O. perennis accessions) also lack the satellite DNA and have a different hybridization pattern with the moderately repeated sequence. All other accessions, irrespective of their geographical origin, are similar. We propose that various accessions of O. perennis from Africa and Asia are closely related to O. sativa and O. glaberrima, and that the dispersal of cultivated and O. perennis rices to different continents may be quite recent. The American O. perennis is a heterogeneous group. Some of the accessions ascribed to this group are closely related to the Asian and African O. perennis, while others have diverged.     

The organisation,nucleotide sequence,and chromosomal distribution of a satellite DNA from Allium cepa

We have investigated the organisation, nucleotide sequence, and chromosomal distribution of a tandemly repeated, satellite DNA from Allium cepa (Liliaceae). The satellite, which constitutes about 4% of the A. cepa genome, may be resolved from main-band DNA in antibiotic-CsCl density gradients, and has a repeat length of about 375 base pairs (bp). A cloned member of the repeat family hybridises exclusively to chromosome telomeres and has a non-random distribution in interphase nuclei. We present the nucleotide sequences of three repeats, which differ at a large number of positions. In addition to arrays made up of 375-bp repeats, homologous sequences are found in units with a greater repeat length. This divergence between repeats reflects the heterogeneity of the satellite determined using other criteria. Possible constraints on the interchromosomal exchange of repeated sequences are discussed.     

Bulb-type onion introgressants posessing Allium fistulosum L. genes recovered from interspecific hybrid backcrosses between A. cepa L. and A. fistulosum L.

Allium fistulosum possesses a number of traits which would be desirable in A. cepa. Thus far, no commercial A. cepa cultivars have been released which harbor Allium fistulosum traits. F₁BC₃ populations were generated for this study by backcrossing A. cepa to A. cepa×A. fistulosum hybrids. The F₁BC₃ plants were evaluated for plant morphology, floral characters, male- sterile cytoplasm, soluble solids and pungency, and isozymes. Overall growth habit and floral characters of the F₁BC₃ plants were much like A. cepa. We report here the recovery of recombinant, bulbing, and fertile A. cepa-type onions that exhibit A. fistulosum isozyme alleles and morphological markers. Recombination between A. cepa and A. fistulosum genomes was achieved using the introgression strategy of backcrossing A. fistulosum into A. cepa, thereby ameliorating the nuclear cytoplasmic barriers that occurred in previous less successful introgression attempts when plants were not in A. cepa cytoplasm. We believe this report to be the first demonstration of onion introgressants that are like A. cepa in appearance, are male- and female-fertile, and possess A. fistulosum genes. Received: 29 May 1999 / Accepted 22 June 1999     

Distribution of a 375 bp repeat sequence inAllium (Alliaceae) as revealed by FISH

Information about evolutionary relationships between species of the genusAllium is desirable in order to facilitate breeding programmes. One approach is to study the distribution of repetitive DNA sequences among species thought on taxonomic grounds, to be closely related. We have used fluorescent in-situ hybridisation (FISH) to examine seven species within sect.Cepa of the genus (A. altaicum, A. cepa, A. fistulosum, A. galanthum, A. pskemense, A. oschaninii andA. vavilovii), one species from sect.Rhizirideum (A. roylei), two species from sect.Allium (A. sativum andA. porrum) and one species from sect.Schoenoprasum (A. schoenoprasum). Each species was probed using a 375 bp repeat sequence isolated fromA. cepa (Barnes & al. 1985), which was generated and labelled by polymerase chain reaction (PCR). No signals were detected in anyAllium species not belonging to sect.Cepa with the exception ofA. roylei, whose designation in sect.Rhizirideum is now questioned. Within sect.Cepa the probe was found to hybridize to the terminal regions of the chromosome arms of all the species examined. In addition a number of interstitial bands were detected. Use of FISH reveals a more detailed map of the location of the repeat sequences than has previously been obtained by C-banding and other staining procedures. The distribution of the terminal and interstitial sites when compared, allow us to identify three species groups namely,A. altaicum andA. fistulosum; A. cepa, A. roylei, A. oschaninii andA. vavilovii; andA. galanthum andA. pskemense.     

Amplification of repeated DNA sequences in wheat X rye hybrids regenerated from tissue culture

Summary Previous C-banding analysis of wheat (Triticum aestivum)X rye (Secale cereale) hybrids regenerated from tissue culture revealed enlarged C-bands in some rye chromosomes, but the molecular nature of the change was not determined. In situ hybridization using two DNA probes containing repeated sequences from rye telomeric heterochromatin was conducted on these wheatX rye hybrids and their progeny to investigate the occurrence of amplification in repeated sequences. Clones pSC 74 and pSC 119, which contain sequences from the 480-bp and 120-bp repeated DNA families of rye, respectively, were used as probes. Amplification of 480-bp repeated sequences in the short arm telomere of chromosome 7R was detected in three wheatxrye hybrids and their progeny. The amplified 480-bp sequences were detected by an enlarged hybridization site for pSC 74 at the 7RS telomere, and by the appearance at this same telomeric site of an unlabeled, blue chromosome segment in an otherwise completely brown chromosome hybridizing entirely to the biotin-labeled pSC 119 probe. This variant form of chromosome 7R was not observed in several Chaupon plants, or in the other hybrids derived from the same embryos, indicating the origin of the change in tissue culture. The amplified sequences were inherited up to at least three generations. Deletions and translocations were also observed.Contribution No. 87-9-J, Kansas Agricultural Experiment Station, Kansas State University     

Wheat specific repetitive DNA sequences — construction and characterization of four different genomic clones

Summary The construction and molecular analysis of four recombinant clones — pTa1, pTa2, pTa7, and pTa8 — is described. The four clones contain different highly repeated sequences of genomic DNA from Triticum aestivum variety Chinese Spring. The wheat specificity has been determined by colony and dot blot hybridization in comparison with total rye DNA (Secale cereale variety Petka). The four clones with a variable degree of specificity were compared by sequence analysis after the recloning of wheat DNA inserts into M13 mp8. Within the sequencing data a tendency can be observed that those repeated sequences which show the highest degree of species specificity contain a significantly increased amount of GC residues.     

Recombinant chromosomes of advanced backcross plants between Allium cepa L. and A. fistulosum L. revealed by in situ hybridization

Cytological analysis of (Allium cepa L.×Allium fistulosum L.)×A. cepa L. F₁BC₃ plants revealed most plants were diploid with 16 chromosomes. Karyotypes of these plants showed recombinant chromosomes. Fluorescence and genomic in situ hybridization patterns of interspecific F₁ hybrid and F₁BC₃ plants revealed A. fistulosum chromosomes or chromosomal segments. A highly repetitive 376-bp DNA sequence and genomic DNA of A. fistulosum revealed similar telomeric hybridization sites when hybridized onto A. fistulosum chromosomes. Cytogenetic evidence showed that A. fistulosum DNA has recombined into the A. cepa genome. Received: 20 October 1999 / Accepted: 11 November 1999     

Genome deterioration: loss of repeated sequences and accumulation of junk DNA

A global survey of microbial genomes reveals a correlation between genome size, repeat content and lifestyle. Free-living bacteria have large genomes with a high content of repeated sequences and self-propagating DNA, such as transposons and bacteriophages. In contrast, obligate intracellular bacteria have small genomes with a low content of repeated sequences and no or few genetic parasites. In extreme cases, such as in the 650kb-genomes of aphid endosymbionts of the genus Buchnera all repeated sequences above 200bp have been eliminated. We speculate that the initial downsizing of the genomes of obligate symbionts and parasites occurred by homologous recombination at repeated genes, leading to the loss of large blocks of DNA as well as to the consumption of repeated sequences. Further sequence elimination in these small genomes seems primarily to result from the accumulation of short deletions within genic sequences. This process may lead to temporary increases in the genomic content of pseudogenes and junk DNA. We discuss causes and long-term consequences of extreme genome size reductions in obligate intracellular bacteria.     

Mobile nucleolus organizing regions (NORs) inAllium (Liliaceae s. lat.)? — Inferences from the specifity of silver staining

NORs and interphase nucleoli have been silver stained inAllium cepa, A. fistulosum, reciprocal crosses between both species, and in different strains of top onions which originated from hybridization betweenA. cepa andA. fistulosum. The variability observed in size, number, and position of active NORs and correspondingly in number (and size) of interphase nucleoli is at least in part strain-specific. These data are taken to indicate that NORs inAllium behave like movable genetic elements.—With respect to the staining specifity of silver nitrate, it was found that AgNO₃ labels (1) nucleoli, (2) NORs (i.e., actively transcribed ribosomal genes) inside the achromatic secondary constrictions, and (3) sometimes (but less pronounced) centromeres; Giemsa banding labels heterochromatin surrounding the NOR but not the nucleolus organizing secondary constriction.     

Conserved and variable regions in the chromosomal and extrachromosomal DNA of halobacteria

Summary Total DNA from Halobacterium halobium and other halobacteria strains is separated into two fractions, FI and FII, which differ in their G+C content. FI DNA, which represents the major part of the genome is highly conserved in all purple-membrane-forming halobacteria. Fraction II (FII) consists in H. halobium of three DNA specimen: (a) the previously isolated plasmid pHH1, (b) a heterogeneous set of ccc-DNA molecules present in the cell in low copies, termed minor-circular DNA (MCD) and (c) a new type of more A-T rich DNA segments (chromosomal islands) which, as described here and by Pfeifer and Betlach (1985), are integrated in FI. Sequences homologous to pHH1 occur only in Halobacterium species closely related to H. halobium (like H. cutirubrum), whereas MCD sequences are present in all purple-membrane-forming halobacteria. The sequences of the newly identified chromosomal islands are only found like pHH1, in Halobacterium species, closely related to H. halobium. Total DNA from square halobacteria exhibits no extended homologies to FI or FII DNA from H. halobium. The only common DNA sequences found in all halobacteria are certain insertion elements (ISH), such as ISH26. Based on these data, halobacteria can be subdivided in at least three major groups.Dedicated to Prof. Dr. F. Lingens to his 60th anniversary     

Molecular characterization of transgenic shallots (Allium cepa L.) by adaptor ligation PCR (AL-PCR) and sequencing of genomic DNA flanking T-DNA borders

Genomic DNA blot hybridization is traditionally used to demonstrate that, via genetic transformation, foreign genes are integrated into host genomes. However, in large genome species, such as Allium cepa L., the use of genomic DNA blot hybridization is pushed towards its limits, because a considerable quantity of DNA is needed to obtain enough genome copies for a clear hybridization pattern. Furthermore, genomic DNA blot hybridization is a time-consuming method. Adaptor ligation PCR (AL-PCR) of genomic DNA flanking T-DNA borders does not have these drawbacks and seems to be an adequate alternative to genomic DNA blot hybridization. Using AL-PCR we proved that T-DNA was integrated into the A. cepa genome of three transgenic lines transformed with Agrobacterium tumefaciens EHA105 (pCAMBIA 1301). The AL-PCR patterns obtained were specific and reproducible for a given transgenic line. The results showed that T-DNA integration took place and gave insight in the number of T-DNA copies present. Comparison of AL-PCR and previously obtained genomic DNA blot hybridization results pointed towards complex T-DNA integration patterns in some of the transgenic plants. After cloning and sequencing the AL-PCR products, the junctions between plant genomic DNA and the T-DNA insert could be analysed in great detail. For example it was shown that upon T-DNA integration a 66bp genomic sequence was deleted, and no filler DNA was inserted. Primers located within the left and right flanking genomic DNA in transgenic shallot plants were used to recover the target site of T-DNA integration.     

Molecular structure of the lampbrush loops nooses of the Y chromosome of Drosophila hydei

The molecular structure of the lampbrush loopforming fertility gene nooses from the short arm of the Y chromosome of Drosophila hydei is described on the basis of cloned DNA sequences which are characteristic for the sequence organization in the lampbrush loop. Y chromosomal lampbrush loops are organized into tandem repeat clusters of loop-specific repetitive DNA sequences and in interspersed repetitive DNA sequences with homologies elsewhere in the genome. In this paper, the basic properties of a repeat unit of the tandemly repeated sequence family ay1 are described. Moreover, it is shown that a loop contains several different domains carrying repeat clusters of the same repeated DNA family but with divergent sequence character. One of these clusters is characterized by an internal duplication of the basic repeat unit. We propose that the tandem repeat DNA family ay1 forms a frame of the lampbrush loop which is required for structural and functional reasons.     

Genome structure of Tetrahymena pyriformiis

Reassociation kinetics of DNA from the macronucleus of the ciliate, Tetrahymena pyriformis GL, has been studied. The genome size determined by the kinetic complexity of DNA was found to be 2.0×10⁸ base pairs (or 1.2×10¹¹ daltons). About 90% of the macronuclear DNA fragments 200–300 nucleotides in length reassociate at a rate corresponding to single-copy nucleotide sequences, and 7–9% at a rate corresponding to moderate repetitive sequences; 3–4% of such DNA fragments reassociate at C₀t practically equal to zero. To investigate the linear distribution of repetitive sequences, DNA fragments of high molecular weight were reassociated and reassociation products were treated with Sl-nuclease. DNA double-stranded fragments were then fractionated by size. It has been established that in the Tetrahymena genome long regions containing more than 2000 nucleotides make up about half of the DNA repetitive sequences. Another half of the DNA repetitive sequences (short DNA regions about 200–300 nucleotides long) intersperse with single-copy sequences about 1,000 nucleotides long. Thus, no more than 15% of the Tetrahymena genome is patterned on the principle of interspersing single-copy and short repetitive sequences. Most of the so called zero time binding or foldback DNA seem to be represented by inverted self-complementary (palindromic) nucleotide sequences. The conclusion has been drawn from the analysis of this fraction isolated preparatively by chromatography. About 75% of the foldback DNA is resistant to Sl-nuclease treatment. The Sl-nuclease resistance is independent of the original DNA concentration. Heat denaturation and renaturation are reversible and show both hyper and hypochromic effects. The majority of the inverted sequences are unique and about 20% are repeated tens of times. According to the equilibrium distribution in CsCl density gradients the average nucleotide content of the palindromic fraction does not differ significantly from that of total macronuclear DNA. It was shown that the largest part of this fraction of the Tetrahymena genome are not fragments of ribosomal genes.     

Nuclear DNA amplification in cultured cells of Oryza sativa L.

Summary Highly repeated nuclear DNA sequences from suspension cultured cells of Oryza sativa L. cv. Roncarolo have been cloned in pBR322. Ten clones with specific digestion patterns have been randomly selected. Nine sequences appear to be organized in a clustered tandem array while one is interpersed in the rice genome. The clones have been used to gather information on: (a) their modulation in cultured cells as compared to whole plant and (b) their distribution in different rice cultivars belonging to the Japonica or Indica subspecies of Oryza sativa L. Hybridization with nuclear DNA isolated either from suspension or from seedlings of the Roncarolo cultivar revealed extensive quantitative variations, with most cloned sequences showing amplification (up to 75-fold) in cultured cells. Hybridization with nuclear DNA isolated from seedlings or suspension cultured cells from different cultivars belonging to the Japonica or to the Indica sub-species of O. sativa have shown that (a) amplification also occurs in a similar pattern in the case of DNA from the other tested suspension cultured cell types but not in the case of DNA from seedlings; (b) in some cases the tested sequences show minor but significant variations in different rice accessions.On leave from China National Rice Research Institute, Hangzhou, China     

Restriction enzyme analysis of the germ line limited DNA of Ascaris suum

The germ line limited DNA of Ascaris suum was isolated from sperm and testis as a satellite DNA component in Hoechst 33258 — CsCl gradients. Employing restriction enzyme analysis, we show that the germ line limited DNA is composed entirely of two families of tandemly repeated sequences, one repeat unit is 125 bp, and the other 131 bp long. The total appr. 5 × 10⁵ copies of the two families are physically separated from each other (segmental arrangement). Several repeat unit variants within both families could be detected. The copies of sequence variants are arranged in tandem (subsegmental arrangement). Reassociation and hybridization experiments revealed similar sequences of the two repeat units. The archaeotypic core sequence of both repeat units is probably a tetranucleotide which shows a theme and variation pattern. During chromatin diminution in the presoma cells the satellite DNA is eliminated from the chromosomes. However, a limited number of tandemly repeated copies of both kinds of repeat units could be detected in the soma genome using radioactive probes of both repeat units in Southern blots of muscle and intestine of adult animals. The tandem arrangement and the hierarchical pattern of restriction sites throughout different subfamilies supports the model of successive segmental amplification events during the evolution of the germ line limited DNA. Since the germ line limited satellite DNA is exclusively located at the ends of the chromosomes, a fold back structure for the telomeric DNA sequences is proposed which might have generated this DNA.     

Ancient repeated sequences in the pea and mung bean genomes and implications for genome evolution

Essentially all of the sequences in the pea (Pisum sativum) genome which reassociate with single copy kinetics at standard (T_m -25°C) criterion follow repetitive kinetics at lower temperatures (about T_m-35°C). Analysis of thermal stability profiles for presumptive single copy duplexes show that they contain substantial mismatch even when formed at standard criterion. Thus most of the sequences in the pea genome which are conventionally defined as single copy are actually fossil repeats — that is, they are members of extensively diverged (mutuated) and thus presumably ancient families of repeated sequences. Coding sequences as represented by a cDNA probe prepared from poly-somal poly(A) + mRNA reassociate with single copy kinetics regardless of criterion and do not form mismatched duplexes. The coding regions thus appear to be composed of true single copy sequences but they cannot represent more than a few percent of the pea genome. Ancient diverged repeats are present, but not a prominent feature of the smaller mung bean (Vigna radiata) genome. An extension of a simple evolutionary model is proposed in which these and other differences in genome organization are considered to reflect different rates of sequence amplification or genome turnover during evolution. The model accounts for some of the differences between typical plant and animal genomes.     

Double-stranded regions in denatured DNA from mouse cells

Approximately 2% of the DNA of the mouse genome reassociates at infinitely low C ₀ t values, 10^-7 to 10^-6 moles 1^-1 s. The melting profile of the reassociation product, which is resistant to nuclease S₁ digestion, has been characterized by hydroxyapatite column chromatography. The properties of these nuclease resistant sequences suggest that they exist as DNA-hairpins and that they originate from reverted base sequences within the genome.     

Genome specific,highly repeated sequences of Hordeum vulgare: cloning,sequencing and squash dot test

Summary Highly repeated sequences of nuclear DNA from barley Hordeum vulgare (L.) variety Erfa were cloned. Several clones containing barley specific repeated DNA were analysed by sequence analysis and Southern blot hybridization. The investigated repeats differ from each other in their length, sequence and redundancy. Their length ranges from 36 bp to about 180 bp. The repeats are AT-rich and differ widely in their redundancy within the barley genome. Southern analysis showed that the repeats belong to different repetition complexes. The possibility for utilizing these clones as probes for simple and fast genome analysis is demonstrated in squash dot experiments.     

Cloning and Analysis of Structural Organization of Hox Genes in the Polychaete Nereis virens

We have undertaken an active search for homeobox-containing sequences of Antpclass (Hoxgenes) in the genome DNA of polychaete Nereis virens. This search was based on the high evolutionary conservation of these sequences, which made possible their amplification in the polymerase chain reaction with degenerate primers. As a result, eleven fragments of various Hoxgenes, including AbdB-like Nvi-post1, were cloned. Using pulsed-field electrophoresis, we have demonstrated that Hoxgenes corresponding to the isolated fragments are clustered in the genome of N. virens.