Isolation of a species-specific satellite DNA with a novel CENP-B-like box from the North African rodent Lemniscomys barbarus.

A species-specific satellite DNA (Lb-MspISAT) was isolated from the North African rodent Lemniscomys barbarus. This DNA is highly homogeneous in the sequence of different repeats and shows no internal repetitions. Filter and in situ hybridizations demonstrated that it is tandemly repeated at the centromeres of all chromosomes of the complement. A 19-bp CENP-B-like motif was found in Lb-MspISAT which conserves 12 of the 17-bp of the human CENP-B box, but only 5 of the 9-bp of the canonical sequence that is necessary to bind the CENP-B protein. Compared with the human CENP-B box, nucleotide substitutions and insertions increase the palindromic structure of this motif. The possibilities that it may be involved in centromeric function or in homogenization of the Lb-MspISAT sequence are discussed.     

Isolation and characterization of satellite DNA from mustard seedlings

The DNA from mustard (Sinapis alba L.) seedlings was examined by neutral CsCl and Ag⁺/Cs₂SO₄ density gradient centrifugation. Different satellite fractions were revealed by these two methods. The satellite fractions obtained from the Ag⁺/Cs₂SO₄ density gradient could not be generally correlated with satellite DNA fractions observed in CsCl. In CsCl density gradient centrifugation, a main band at density 1,695 g/cm³ and a heavy shoulder at density 1,703 g/cm³ are found. By preparative CsCl gradient centrifugation the heavy shoulder can be enriched but not completely separated from the main band DNA.—Gradient centrifugation by complexing the DNA with Ag⁺ rf. 0.25 to DNA phosphate reveals three distinct fractions which are further characterized: The heavy satelite DNA fraction revealed by Ag⁺/Cs₂SO₄ gradient centrifugation has the same density in a CsCl gradient and the same Tm value as the main band, but differs from main band DNA in the details of its melting profile and in its renaturation kinetics. The light Ag⁺/Cs₂SO₄ satellite DNA fraction had a higher melting temperature corresponding to a GC-rich base composition. Differences between these 3 fractions are observed in thermal denaturation and renaturation profiles, hybridization in situ with ribosomal RNA, and their response to restriction endonuclease digestion. The light satellite fraction from the Ag⁺/Cs₂SO₄ gradient, rich in ribosomal cistrons corresponds to the heavy shoulder DNA of neutral CsCl gradients which also is rich in ribosomal cistrons. The heavy satellite fraction from Ag⁺/Cs₂SO₄ gradient which contains highly repetitive short nucleotide sequences could not be revealed by the classical CsCl gradient centrifugation technique.     

Isolation and characterization of a linear DNA plasmid from Streptomyces clavuligerus

Summary A linear DNA plasmid (pSCL) has been isolated from Streptomyces clavuligerus by a method employing high concentrations of protease. Rate-zonal sedimentation on sucrose gradients was used to purify the plasmid. The plasmid is 12 kb in length and appears to be linked to protein at its 5 termini. A restriction endonuclease map of the plasmid for ten enzymes has been determined. Evidence for terminally repeated sequences is provided by cross-hybridization analysis.     

Nomenclatural changes resulting from the transfer of tropical African Sarcostemma to Cynanchum ( Apocynaceae : Asclepiadoideae )

Summary  Four species of tropical African Sarcostemma are transferred to Cynanchum together with two subspecies of S. viminale. In addition, Sarcostemma mulanjense is reduced to subspecific rank under C. viminale.     

Isolation and Characterization of a Satellite DNA Family in Achirus lineatus (Teleostei: Pleuronectiformes: Achiridae)

Agarose gels stained with Ethidium bromide and Southern blot experiments of HindIII-digested genomic DNA of Achirus lineatus evidenced the presence of monomers and multimers of a DNA segment of about 200 bp, named here Al-HindIII sequence. No signals were observed in Southern blot experiments with genomic DNA of other flatfish species. The DNA sequencing of four recombinant clones showed that Al-HindIII sequences had 204 bp and were 63.72% AT-rich. FISH experiments using a Al-HindIII sequence as probe showed bright signals in the centromeric position of all chromosomes of A. lineatus.     

Isolation of a DNA polymerase I (polA) mutant of Rhizobium leguminosarum that has significantly reduced levels of an IncQ-group plasmid

A population of Tn5 mutagenised Rhizobium leguminosarum cells was screened for mutants affected in protein secretion by introducing a plasmid carrying the Erwinia chrysanthemi prtB gene and screening for mutants defective in secretion of the protease PrtB. One such mutant (A301) also appeared to be defective in secretion of the R. leguminosarum nodulation protein NodO. Genetic analysis showed that the defect in A301 was caused by the Tn5 insertion. However the DNA sequence adjacent to the site of Tn5 insertion had significant homology to the Escherichia coli polA gene, which encodes DNA polymerase I. The mutant A301 showed increased sensitivity to ultraviolet light, a characteristic of polA mutants of E. coli. The apparent defect in secretion by A301 was due to a large decrease in the copy number of the IncQ group replicon on which prtB and nodO were cloned and this decreased the total amounts of PrtB or NodO protein synthesised and secreted by the polA mutant. The polA mutant had a lower growth rate than the parent strain on both rich and minimal media, but there was no obvious effect of the polA mutation on the symbiosis of R. leguminosarum bv. viciae with pea.     

Isolation, characterization and molecular cloning of new temporins from the skin of the North African ranid Pelophylax saharica

Temporins are small antimicrobial peptides isolated from North American and Eurasian ranid frogs that are particularly active against Gram-positive bacteria. To date, no temporins have been characterized from North African frog species. We isolated three novel members of the temporin family, named temporin-1Sa (FLSGIVGMLGKLF(amide)), -1Sb (FLPIVTNLLSGLL(amide)), and -1Sc (FLSHIAGFLSNLF(amide)), from the skin of the Sahara frog Pelophylax (Rana) saharica originating from Tunisia. These temporins were identified by a combined mass spectrometry/molecular cloning approach. Temporin-1Sa was found to be highly active against Gram-positive and Gram-negative bacteria, yeasts and fungi (MIC=2-30muM). To our knowledge, this is the first 13-residue member of the temporin family with a net charge of +2 that shows such broad-spectrum activity with particularly high potency on the clinically relevant Gram-negative strains, Escherichia coli (MIC=10muM) and Pseudomonas aeruginosa (MIC=31muM). Moreover, temporin-1Sa displays significant antiparasitic activity (IC(50) approximately 20muM) against the promastigote and amastigote stages of Leishmania infantum.     

Isolation and characterization of genome-specific DNA sequences in Triticeae species

Two contrasting genome-specific DNA sequences were isolated from Aegilops speltoides (wild goat grass) and Hordeum chilense (wild barley), each representing more than 1 % of the genomes. These repetitive DNA fragments were identified as being genome-specific before cloning by genomic Southern hybridization (using total genomic DNA as a probe), and hence extensive screening of clones was not required. For each fragment, up to six recombinant plasmid clones were screened and about half were genome-specific. Clone pAesKB52 from Ae. speltoides was a 763 by EcoRI fragment, physically organized in simple tandem repeats and shown to localize to sub-telomerec chromosome regions of species with the Triticeae S-genome by in situ hybridization to chromosomes. The sequence data showed an internal duplication of some 280 bp, which presumably occurred before sequence amplification and dispersion, perhaps by unequal crossing-over or reciprocal translocation. In situ hybridization showed that the sequence distribution varied between closely related (S-genome) species. Clone pHcKB6 was a 339 by DraI fragment from H. chilense, also tandemly repeated but more variable; loss of the DraI site resulting in a ladder pattern in Southern blots which had little background smear. In situ hybridization showed that the tandem repeats were present as small clusters dispersed along all chromosome arms except at a few discrete regions including the centromeres and telomeres. The clone hybridized essentially specifically to the H-genome of H. chilense and hence was able to identify the origin of chromosomes in a H. chilense x Secale africanum hybrid by in situ hybridization. It has a high A + T content (66%), small internal duplications, and a 50 by degenerate inverted repeat. We speculate that it has dispersed by retrotransposition in association with other sequences carrying coding domains. The organization and evolution of such sequences are important in understanding long-range genome organization and the types of change that can occur on evolutionary and plant breeding timescales. Genome-specific sequences are also useful as markers for alien chromatin in plant breeding.     

Domoic acid production by Pseudo-nitzschia australis and Pseudo-nitzschia calliantha isolated from North Chile

The morphology and toxicity of the four ubiquitous species belonging to the genus Pseudo-nitzschia found in mixed blooms of phytoplankton from northern Chilean waters were studied. The phytoplankton samples and cultures obtained were identified by scanning electron microscopy, revealing the presence of Pseudo-nitzschia australis, P. calliantha, P. pseudodelicatissima and P. subfraudulenta. This is the first report of P. calliantha in northern Chile. Toxin analyses using the LC–MS method confirmed the presence of domoic acid in P. australis and P. calliantha. Domoic acid was not detected in cultures of P. subfraudulenta. This study therefore confirms P. australis and P. calliantha as an unequivocal source of domoic acid in Chilean waters. P. australis is probably the most important producer of amnesic shellfish toxin in view of its domoic acid content. However, more research is needed to evaluate the potential for toxin production in P. pseudodelicatissima.     

Characterization of a New HpaI Centromeric Satellite DNA in Salmo salar

A highly repeated HpaI DNA family was revealed in Atlantic salmon (Salmo salar) and analyzed by Southern blotting and fluorescence in situ hybridization (FISH). In this report, we describe the nucleotide sequence, genomic structure and chromosomal localization of this HpaI repeat. This novel satellite appeared tandemly arrayed and located at centromeric areas of three acrocentric chromosome pairs as evidenced by FISH. The sequence was characterized by a high AT content (63%), a short consensus motif (A/T)(G/C)AAA(T/C) similar to other centromeric satellites motifs, and by short AT enriched stretches. The presence of this sequence in other salmonid species was also tested by Southern blot hybridization and used to analyze its evolution within this group.     

A highly repeated DNA sequence in Arabidopsis thaliana

Summary Three members of a family of highly repeated DNA sequences from Arabidopsis thaliana have been cloned and characterized. The repeat unit has an average length of 180 bp and is tandemly repeated in arrays longer than 50 kb. This family represents more than one percent of the Arabidopsis genome. Sequence comparisons with tandemly repeated DNA sequences from other Cruciferae species show several regions of homology and a similar length of the repeat unit. Homologies are also found to highly repeated sequences from other plant species. When the sequence CCGG occurs in the repeated DNA, the inner cytosine is generally methylated.     

Petunia plants escape from negative selection against a transgene by silencing the foreign DNA via methylation

Summary TransgenicPetunia hybrida clones harbouring the T-DNA gene2 ofAgrobacterium tumefaciens were used to test a strategy for the trapping of plant transposable elements. In thePetunia line used, floral variegation is due to the presence of the non-autonomous transposable elementdTph1 at theAn1 locus. The gene2 product converts the auxin precursor indole-3-acetamide and its analogue 1-naphthalene acetamide into the active auxins indole-3-acetic acid and 1-naphthalene acetic acid. Plant cells that express gene2 can use a low concentration of the precursors as auxins and become sensitive to the toxicity of high concentrations of these compounds. By selecting protoplast-derived microcalli or seedlings able to grow on medium with high precursor concentrations, variant plants were obtained in which gene2 was no longer expressed. Southern analysis, using gene2-specific probes, revealed that in one variant the T-DNA was deleted. For 30 other variants no alteration in gene2 structure was observed, indicating that transposable element insertion was not responsible for the inactivation of gene2. Analysis with restriction enzymes allowing discrimination between methylated or non-methylated DNA sequences showed that the inactivated gene2 sequences were methylated. Addition of the in vivo methylation inhibitor 5-azacytidine to the medium led to reactivation of gene2 expression in some of the variants. These observations demonstrated that reversible DNA methylation was the main cause of silencing of gene2 in this system.     

Isolation of genomic DNA from the earthworm speciesEisenia fetida

Our interest in detecting genotoxic exposure in earthworms led us to isolate high quality DNA from theEisenia fetida species. For that, we compared a modification of the conventional phenol-chloroform extraction procedure, usually refered to as the Maniatis procedure, to two commercially available kits reportedly eliminating multiple partitions in phenol and chloroform, namely the Qiagen and Nucleon protocols. From the 260 nm optical density values, the commercial kits extracts hinted toward higher DNA recovery with those procedures. However, the 260/280 nm ratios indicated that the quality of the DNA isolated with the modified Maniatis procedure was purer than that isolated with the commercial kits, the latter being most probably contaminated by proteins and/or RNA. The Maniatis procedure was slightly modified by the introduction of a potassium acetate step for protein precipitation and by shortening the proteinase K treatment from 12–18 h to only 2 h. The higher quality of the DNA isolated by phenol-chloroform extraction was confirmed by quantification with the fluorescent 3,5-diaminobenzoic acid assay. Preliminary results suggest that the modified Maniatis procedure herein described is not only applicable for DNA adducts studies using³²P-postlabelling techniques but is also suitable for DNA extraction from other earthworm species such asLumbricus terrestris.     

Isolation of DNA from plants high in polyphenolics

DNA suitable for digestion with restriction endonucleases has been isolated fromTheobroma cacao, a plant high in polyphenolic compounds, and several other plant taxa using the method presented in this paper. The method relies on concentrating nuclei away from most of the cytoplasmic components and inhibiting the formation of oxidized polyphenolic compounds and their interaction with the DNA fraction in the remaining steps.     

DNA supercoiling in a thermotolerant mutant ofEscherichia coli

A spontaneously occurring, nalidixic acid-resistant (Nal^R), thermotolerant (T/r) mutant ofEscherichia coli was isolated. Bacteriophage P1-mediated transduction showed that Nal^R mapped at or neargyr A, one of the two genes encoding DNA gyrase. Expression ofgyrA ⁺ from a plasmid rendered the mutant sensitive to nalidixic acid and to high temperature, the result expected for alleles mapping ingyrA. Plasmid linking number measurements, made with DNA from cells grown at 37° C or shifted to 48° C, revealed that supercoiling was about 12% less negative in the T/r mutant than in the parental strain. Each strain preferentially expressed two different proteins at 48° C. The genetic and supercoiling data indicate that thermo-tolerance can arise from an alteration in DNA gyrase that lowers supercoiling. This eubacterial study, when. coupled with those of archaebacteria, suggests that DNA relaxation is a general aspect of thermotolerance.     

Nucleotide sequence of the recF gene cluster from Staphylococcus aureus and complementation analysis in Bacillus subtilis recF mutants

We have determined the nucleotide sequence of a 3.5 kb segment in the recF region of the Staphylococcus aureus chromosome. The gene order at this locus, dnaA-dnaN-recF-gyrB is similar to that found in the replication origin region of many other bacteria. S. aureus RecF protein (predicted molecular mass 42.3 kDa), has 57% amino acid sequence identity with the Bacillus subtilis RecF protein (42.2 kDa), but only 26% with the Escherichia coli RecF protein (40.5 kDa). We have shown that the S. aureus recF gene partially complements the defect of a B. subtilis recF mutant, but does not complement an E. coli recF strain. The amino acid sequence alignment of seven available RecF proteins (five of them from bacteria of gram-negative origin) allowed us to identify eight highly conserved regions ( to ) and to predict five new conserved regions within the gram-positive group (a to f). We suggest that the basic mechanism of homologous recombination is conserved among free-living bacteria.     

The spacing of Escherichia coli DNA gyrase sites cleaved in vivo by treatment with oxolinic acid and sodium dodecyl sulfate

In an attempt to locate gyrase binding sites in a specific region of the chromosome of E. coli, we have reinvestigated gyrase-promoted cleavage of chromosomal DNA by oxolinic acid and sodium dodecyl sulfate. Contrary to a previous report suggesting the presence of one site every 100 kb of DNA (Snyder and Drlica, J. Mol. Biol. 131, 287-302), we found frequencies of one cleavage every 25 or 12 kb depending on the growth medium. A search for cleavage sites by Southern blot hybridization failed to reveal any binding site cleaved at a high frequency. These results suggest that the actual spacing of sites is much closer than that determined from the frequency of cleavage. Measurement of the average size of fragments containing defined DNA sequences indicated that the frequency of sites varies along the chromosome. The region located opposite to oriC carries relatively few sites.     

Reduced Rates of Sequence Evolution of Y-Linked Satellite DNA in Rumex (Polygonaceae)

One characteristic of sex chromosomes is the accumulation of a set of different types of repetitive DNA sequences in the Y chromosomes. However, little is known about how this occurs or about how the absence of recombination affects the subsequent evolutionary fate of the repetitive sequences in the Y chromosome. Here we compare the evolutionary pathways leading to the appearance of three different families of satellite-DNA sequences within the genomes of Rumex acetosa and R. papillaris, two dioecious plant species with a complex XX/XY₁Y₂ sex-chromosome system. We have found that two of these families, one autosomic (the RAE730 family) and one Y-linked (the RAYSI family), arose independently from the ancestral duplication of the same 120-bp repeat unit. Conversely, a comparative analysis of the three satellite-DNA families reveals no evolutionary relationships between these two and the third, RAE180, also located in the Y chromosomes. However, we have demonstrated that, regardless of the mechanisms that gave rise to these families, satellite-DNA sequences have different evolutionary fates according to their location in different types of chromosomes. Specifically, those in the Y chromosomes have evolved at half the rate of those in the autosomes, our results supporting the hypothesis that satellite DNAs in nonrecombining Y chromosomes undergo lower rates of sequence evolution and homogenization than do satellite DNAs in autosomes.[Reviewing Editor: DR. Jerzy Jurka]     

Isolation of elatol from Laurencia microcladia and its palatability to the sea urchin Echinometra lucunter

Elatol was isolated as the major compound from the red alga Laurencia microcladia Kütz. collected in Southern Brazil. This is the first report of elatol in this species. We also investigated the herbivore behaviour of the black sea urchin Echinometra lucunter (Linnaeus, 1758) towards L. microcladia, Ulva fasciata Delile and Gracilaria domingensis (Kütz.) Sond. ex Dickie through live algal multiple-choice feeding assay, as well as artificial feeding assay. The sea urchins ate the crude algae L. microcladia and pellets containing the powdered algae, extract and all tested concentrations of elatol, suggesting that this seaweed and its main compound are palatable for E. lucunter.