Is repetitive DNA involved in male gametogenesis ofNarcissus papyraceus?

Summary The male gametophyte of the Spermatophyta is of particular interest when studying quantitative modifications of repetitive DNA which accompany plant differentiation. In fact, this well documented system offers the opportunity to investigate the phenomenon at the nuclear level. In particular, it permits comparison between single haploid nuclei which, though derived from the same mitotic division, have different, well-defined morphological and functional processes. The aim of the present work was to investigate the behaviour of repetitive DNA during male gametogenesis inNarcissus papyraceus. By the use of A+T and G+C specific fluorochromes (DAPI and Chromomycin A₃), and by in situ DNA digestion with appropriate restriction endonucleases (Eco RI and Hae III), it is shown that during the course of gametogenesis, generative and vegetative nuclei behave differently with respect to particular sections of the genome. Characteristic agglomerations of specific A+T rich DNA sequences can be observed, localized by the two distal ends of the spindle-shaped generative nucleus. The surface area occupied by the agglomerations differs significantly at the ends in all observed cases. The above agglomerations are present in most of the generative nuclei and are probably composed of repetitive DNA sequences. Some embryological questions are discussed in the light of these results.Abbreviations DAPI 4,6-diamidino-2-phenylindole - Eco RI Eco RI with star activity (N/AATTN) Dedicated to Prof. E. Battaglia on the occasion of his 75th birthday     

A novel repetitive DNA sequence in the genus Oryza

Summary Repetitive DNA sequences in the genus Oryza (rice) represent a large fraction of the nuclear DNA. The isolation and characterization of major repetitive DNA sequences will lead to a better understanding of rice genome organization and evolution. Here we report the characterization of a novel repetitive sequence, CC-1, from the CC genome. This repetitive sequence is present as long tandem arrays with a repeat unit 194 bp in length in the CC-diploid genome but 172 bp in length in the BBCC and CCDD tetraploid genomes. This repetitive sequence is also present, though at lower copy numbers, in the AA and BB genomes, but is absent in the EE and FF genomes. Hybridization experiments revealed considerable differences both in copy numbers and in restriction fragment patterns of CC-1 both between and within rice species. The results support the hypothesis that the CC genome is more closely related to the AA genome than to the BB genome, and most distantly related to the EE and FF genomes.     

Molecular and cytogenetic characterization of repetitive DNA sequences from Lolium and Festuca: applications in the analysis of Festulolium hybrids

Summary A set of species-specific repetitive DNA sequences was isolated from Lolium multiflorum and Festuca arundinacea. The degree of their species specificity as well as possible homologies among them were determined by dot-blot hybridization analysis. In order to understand the genomic organization of representative Lolium and Festuca-specific repetitive DNA sequences, we performed Southern blot hybridization and in situ hybridization to metaphase chromosomes.Southern blot hybridization analysis of eight different repetitive DNA sequences of L. multiflorum and one of F. arundinacea indicated either tandem and clustered arrangements of partially dispersed localization in their respective genomes. Some of these sequences, e.g. LMB3, showed a similar genomic organization in F. arundinacea and F. pratensis, but a slightly different organization and degree of redundancy in L. multiflorum. Clones sequences varied in size between 100 bp and 1.2 kb. Estimated copy number in the corresponding haploid genomes varied between 300 and 2×10⁴. Sequence analysis of the highly species-specific sequences from plasmids pLMH2 and pLMB4 (L. multiflorum specific) and from pFAH1 (F. arundinacea specific) revealed some internal repeats without higher order. No homologies between the sequences or to other repetitive sequences were observed. In situ hybridization with these latter sequences to metaphase chromosomes from L. multiflorum, F. arundinacea and from symmetric sexual Festulolium hybrid revealed their relatively even distribution in the corresponding genomes. The in situ hybridization thus also allowed a clearcut simple identification of parental chromosomes in the Festulolium hybrid.The potential use of these species-specific clones as hybridization probes in quantitative dot-blot analysis of the genomic make-up of Festulolium (sexual and somatic) hybrids is also demonstrated.Abbreviations bp Base pair (s) - CMA chromomycin A₃ - DAPI 4,6-diamidino-2-phenylindole - IPTG isopropyl -D-thio-galactopyranoside - kb kilobase pair(s) - NBT nitroblue tetrazolium chloride - X-gal 5-bromo-4-chloro-3-inonyl -D-galactopyranoside     

Agar Analysis,nuclear genome quantification and characterization of four agarophytes (Gracilaria) from the Mexican Gulf Coast

DNA reassociation kinetics were used to determine nuclear genome organization and complexity in four species of Gracilaria (Gracilariales, Rhodophyta). In Gracilaria tikvahiae, G. caudata, G. cervicornis and G. divaricata, results indicate the presence of three second order components corresponding to fast, intermediate and slow fractions. Repetitive sequences varied from 13–46% and unique DNA ranged from 45–78%, Thermal denaturation (T _m) indicated guanine + cytosine (G + C) levels of 41.9–46.0 mol % G + C. Microspectrophotometry with the DNA-localizing fluorochrome DAPI was used to quantify nuclear DNA content. Comparisons of mean nuclear DNA (I _f) values to chicken erythrocytes (RBC) resulted in an estimate of 0.37–0.40 pg/2C genomes for the four Gracilaria species. Total agar content following alkaline pretreatment ranged from 7–15% dry weight. Gel strengths were generally below commercial levels, ranging from 40–260 g cm⁻² Nuclear genome profiles developed from information 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 as previously speculated.     

Molecular analysis of the recombination junctions of lambda bio transducing phages.

Summary To examine the mechanism of recombination involved in the formation of specialized transducing phage during the induction of bacteriophage we have determined the nucleotide sequences of the recombination junctions of bio phages. The results indicate that abnormal excision takes place at many sites on both bacterial and phage genomes and that the recombination sites have short regions of homology (5–14 bp). Some of the sequences of the recombination sites were similar to the consensus sequences of DNA gyrase-cleavage sites and repetitive extragenic palindromic (REP) sequences. These results showed that abnormal excision is a type of illegitimate recombination. The possible involvement of DNA gyrase in this recombination is discussed.     

The distribution and divergence during evolution of families of repetitive DNA sequences inLathyrus species

Summary Evolution and divergence among, species within the genusLathyrus have involved an approximately fivefold increase in the amounts of nuclear DNA. Most species inLathyrus are diploids with the same chromosome number, 2n=14. Significant changes in the amounts of repetitive sequences have accounted for much of the evolutionary DNA variation between species. Seven diploidLathyrus species with a twofold variation in nuclear DNA amounts between them were investigated. Using higher derivative analysis of the thermal denaturation profiles of the reassociated repetitive DNA, the reiteration frequency and divergence of repetitive families were compared. Much variation in the reiteration frequency was observed within and between species. In species with larger 2C DNA amounts repetitive families had on average greater amounts of DNA. Despite the massive differences in DNA amounts, six species were consistently similar in the number of repetitive families in their genomes, and they showed a similar pattern in base sequence divergence. In terms of base sequence relationships the repetitive families appeared to be heterogeneous. The evolutionary significance is discussed.     

Interspecific “common” repetitive DNA sequences in salamanders of the genus Plethodon

Intermediate repetitive sequences of Plethodon cinereus which comprised about 30% of the genomic DNA were isolated and iodinated with ¹²⁵I. About 5% of the ¹²⁵I-repetitive fraction hybridized with a large excess of DNA from P. dunni at Cot 20. About half of the ¹²⁵I-DNA in the hybrids was resistant to extensive digestion with S-1 nuclease. The average molecular size of the S-1 nuclease-resistant fraction was about 100 nucleotide pairs. The melting temperature of the S-1 nuclease-resistant fraction was about 2° lower than that of the corresponding fraction made with P. cinereus DNA. These results are taken to indicate the presence in the genomes of P. cinereus and P. dunni of evolutionarily stable common repetitive sequences. The average frequency of repetition of the common repetitive sequences is about 6,000 × in both species. The common repetitive fraction is also present in the genomes of other species of Plethodon, although the general populations of intermediate repetitive sequences are markedly different from one species to another. The cinereus-dunni common repetitive sequences could not be detected in plethodontids belonging to different tribes, nor in more distantly related amphibians. The profiles of binding of the common repetitive sequences to CsCl or Cs₂SO₄-Ag⁺ density gradient fractions of P. dunni DNA suggested that these sequences consisted of heterogeneous components with respect to base compositions, and that they did not include large amounts of the genes for ribosomal RNA, 5S RNA, 4S RNA, or histone messenger RNA. — In situ hybridization of the ³H-labelled intermediate repetitive sequences of P. cinereus to male meiotic chromosomes of the same species gave autoradiographs after an exposure of seven days showing all 14 chromosomes labelled. The pattern of labelling appeared not to be random, but was impossible to analyse on account of the irregular shapes and different degrees of stretching of diplotene and prometaphase chromosomes. In situ hybridization of the same sequences to meiotic chromosomes from P. dunni gave autoradiographs after 60 d exposure in which all chromosomes were labelled. These heterologous in situ hybrids can only have involved the common repetitive sequences.     

Genome structure and divergence of nucleotide sequences in echinodermata

The arrangement of repetitive and single-copy DNA sequences has been studied in DNA of some species of Echinodermata — sea urchin, starfishes and sea-cucumber. Comparison of the reassociation kinetics of short and long DNA fragments indicates that the pattern of DNA sequence organization of all these species is similar to the so called Xenopus pattern characteristic of the genomes of most animals and plants. However, substantional variations have been found in the amount of repetitive nucleotide sequences in DNA of different species and in the length of DNA regions containing adjacent single-copy and repetitive sequences. Measurements of the size of S₁-nuclease resistant reassociated repetitive DNA sequences show a variability of ratios between long and short repetitive DNA sequences of different species. — The degree of divergence of short and long repetitive DNA sequences and single-copy DNA was studied by molecular hybridization of the sea urchin Strongylocentrotus intermedius ³H-DNA with the DNA of other species and by determination of the thermostability of the hybridized molecules so obtained. All three fractions of S. intermedius DNA contain sequences homologous to DNA of the other echinoderm species studied. The results obtained suggest that short repetitive DNA sequences are those which have been most highly conserved throughout the evolution of Echinodermata. A new hypothesis is proposed to explain the nature of the evolutionary changes in DNA sequence interspersion patterns.     

Evidence for the wide distribution of repetitive DNA sequences in the genusStreptomyces

Summary Repeated DNA sequences were detected as rapidly reannealing sequences in the chromosomal DNA of 13 out of 14Streptomyces species using either hypochromicity measurements or hydroxyapatite chromatography. These sequences made up between approximately 4% and 11% of the total DNA of these species; only inStreptomyces rimosus were repeated DNA sequences not detected. The repeated sequences fall into a number of distinct percentage G+C (%G+C) classes, many being of rather low %G+C. Analytical density ultracentrifugation of the DNA of these species indicated satellite bands of low %G+C, and high-resolution thermal denaturation profiles indicated the presence of blocks of DNA of low G+C content too. No such satellite band could be found inStreptomyces coelicolor and no low-%G+C DNA could be detected in its thermal denaturation profile. The possible relationship of this repeated DNA, an unusual occurrence in a procaryote, to genetic instability and genetic control mechanisms inStreptomyces is discussed.     

Cloning and characterization of G+C-rich, highly repeated DNA sequences from Porphyra yezoensis (laver), Rhodophyta

G+C-rich sequences in the genomic DNA of Porphyrayezoensis (laver) were cloned and characterized. Sequence analyses of the genomic DNA inserted in fiveclones showed that the DNA contained long G+C-richstretches of more than 200 bp. These stretchesconsisted of more than 80% G+C residues. TheG+C-rich sequences were highly repeated andinterspersed throughout the genome of P.yezoensis and constituted about 6.0–6.6% of thegenome. Parts of these sequences were tandem repeatedin arrays. Hybridization experiments showed thatthese highly repeated, interspersed G+C-rich sequenceswere present in other species of Porphyra, butnot in species of the genera Grateloupia and Gelidium, suggesting that these sequences mightevolve rapidly among genomes, species and genera.     

Hypomethylated sequences: Characterization of the duplicate soybean genome

Soybean is believed to be a diploidized tetraploid generated from an allotetraploid ancestor. In this study, we used hypomethylated genomic DNA as a source of probes to investigate the genomic structure and methylation patterns of duplicated sequences. Forty-five genomic clones from Phaseolus vulgaris and 664 genomic clones from Glycine max were used to examine the duplicated regions in the soybean genome. Southern analysis of genomic DNA using probes from both sources revealed that greater than 15% of the hypomethylated genomic regions were only present once in the soybean genome. The remaining ca. 85% of the hypomethylated regions comprise duplicated or middle repetitive DNA sequences. If only the ratio of single to duplicate probe patterns is considered, it appears that 25% of the single-copy sequences have been lost. By using a subset of probes that only detected duplicated sequences, we examined the methylation status of the homeologous genomes with the restriction enzymes MspI and HpaII. We found that in all cases both copies of these regions were hypomethylated, although there were examples of low-level methylation. It appears that duplicate sequences are being eliminated in the diploidization process. Our data reveal no evidence that duplicated sequences are being silenced by inactivation correlated with methylation patterns.     

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     

Novel methylation at GpC dinucleotide in the fish Sparus aurata genome

To date, vertebrate DNA has been found methylated at the 5 position of cytosine exclusively in dinucleotide CpG or CpNpG stretches. On the the other hand, we determined that cytosine was methylated unusually in dinucleotide GpC at 5-GGCC-3 sequences in the teleost Sparus aurata EcoRI satellite DNA family. This finding is the first example of methylated GpC sequences in the eukaryotic genomes. At this regard, we have examined the relative methylation levels at this site of the highly repetitive EcoRI satellite DNA family from Sparus aurata different tissues. The EcoRI repeat was remarkably more methylated in male germ cells but hypomethylated in female germ cells at the Hae III restriction site ( GpC). The novel modification and the differential methylation pattern suggest that EcoRI satellite could have a structural and/or functional role at the centromeres of Sparus aurata.     

Changes in DNA composition in the evolution of Vicia species

Summary The composition of nuclear DNA in 3 Vicia species are compared. The species V. eriocarpa, V. johannis and V. melanops are from three separate subgeneric sections of Vicia and show a fourfold variation in their amounts of nuclear DNA. DNA melting experiments, buoyant density gradient analysis and Cot reassociation experiments show that the quantitiative change in nuclear DNA between the three species is achieved by changes in the amounts of both repetitive and nonrepetitive DNA sequences. It is suggested that while the increase in the repetitive fraction is achieved by the proliferation of repetitive base sequences the increase in the nonrepetitive fraction is due to the steady accretion of highly diverged base sequences resulting from mutations, deletions, insertions and base sequence rearrangements among families of repetitive sequences.     

Quantitative variation of “Mus musculus-like” constitutive heterochromatin and satellite DNA-sequences in the genus Mus

The extent of conservation of constitutive heterochromatin in three species of Mus viz. M. musculus, M. booduga and M. dunni, with shared cytological properties and homologous DNA sequences has been studied. The cytological properties were investigated by doing fluorescence staining and condensation inhibition of their chromosomes with Hoechst 33258. Both the parameters indicate the occurrence of a reduced quantum of M. musculus like heterochromatin at specific sites in the other two genomes. In situ hybridization of the nick translated ³H-labelled M. musculus satellite DNA with M. booduga and M. dunni chromosomes, also corroborates our Hoechst 33258 findings and comparable variation in the amount and site of occurrence of sequences homologous to M. musculus satellite DNA in these species are noticed. The study thus provides a good example of a gradual quantitative variation of a particular type of heterochromatin and in turn of the repetitive DNA constituting it in different related species. Further since the heterochromatin in M. booduga and M. dunni is expected to contain different repetitive DNA sequences in addition to those homologous to M. musculus satellite DNA, it is proposed that a change in the balance between two or more repetitive sequences in heterochromatin may be more crucial in its evolutionary consequences rather than a mere increase or decrease of a homogeneous repetitive sequence.     

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.     

Unique repetitive sequences of 170 bp inChlorella

Summary Cot analysis ofChlorella DNA revealed that the genome of the unicellular green alga contained a small amount of repetitive sequences (at most 15% of the total DNA). Short repetitive sequences (SRS) of 170 bp produced by enzymatic digestion of algal DNA with eitherHaeIII,HinfI, orPstI, were found by polyacrylamide gel electrophoresis, and their copy number was estimated to be a few hundred (about 2% of the total repetitive sequences). All three members showed high sequence homology and could be be unified into one family, HaeIII family. The family was divided further into two subfamilies,HinfI- (HaeIII-andHinfI-SRS) andPstI-(PstI-SRS) subfamilies, based on small sequence differences among the members. TheHaeIII family had characteristic structural features, including a considerable number of small unique sequence units (purine-CC) and both direct and inverted repeats, and were organized in tandem arrays in the genome.     

Purification and characterization of two transcribed repetitive DNA fractions from the pigeon genome

Two fractions of the repeats belonging to intermediate frequency repetitive DNA were isolated from the total pigeon nuclear DNA fragmented to about 450 nucleotides. One fraction was designated as rare repeats (repetition frequency about 35 per haploid genome) and another termed as moderate repeats (repetition frequency about 2500 per haploid genome). The rare repeats, which constitute about 7% of the total DNA, include at least 75% of the repetitive DNA sequences transcribed into the high molecular fraction (>45S) of HnRNA in erythroid cells. These repeats have properties compatible with the characteristics of the class of low frequency interspersed DNA found in genomes of many other Metazoan species. The moderate repeats contribute only about 10–20% of the total repetitive DNA copies present in >45S HnRNA and differ from the rare repeats in some other properties. — The possible role of the rare repeats in the genome is discussed.     

DNA sequence organization in the genome of Nicotiana tabacum

The genome of Nicotiana tabacum was investigated by DNA/DNA reassociation for its spectrum of DNA repetition components and pattern of DNA sequence organization. The reassociation of 300 nucleotide DNA fragments analyzed by hydroxyapatite chromatography reveals the presence of three major classes of DNA differing in reiteration frequency. Each class of DNA was isolated and characterized with respect to kinetic homogeneity and thermal properties on melting. These measurements demonstrate that the genome of N. tabacum has a 1C DNA content of 1.65 pg and that DNA sequences are represented an average of 12,400, 252, and 1 times each. — The organization of the DNA sequences in the N. tabacum genome was determined from the reassociation kinetics of long DNA fragments as well as S1 nuclease resistance and hyperchromicity measurements on DNA fragments after annealing to C₀t values at which only repetitive DNA sequences will reassociate. At least 55% of the total DNA sequences are organized in a short period interspersion pattern consisting of an alternation of single copy sequences, averaging 1400 nucleotides, with short repetitive elements approximately 300 nucleotides in length. Another 25% of the genome contains long repetitive DNA sequences having a minimal genomic length of 1500 nucleotides. These repetitive DNA sequences are much less divergent than the short interspersed DNA sequence elements. These results indicate that the pattern of DNA sequence organization in the tobacco genome bears remarkable similarity to that found in the genomes of most animal species investigated to date.