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 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 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.     

Novel hosts of the Eucalyptus canker pathogen Chrysoporthe cubensis and a new Chrysoporthe species from Colombia

The pathogen Chrysoporthe cubensis (formerly Cryphonectria cubensis) is best known for the important canker disease that it causes on Eucalyptus species. This fungus is also a pathogen of Syzygium aromaticum (clove), which is native to Indonesia, and like Eucalyptus, is a member of Myrtaceae. Furthermore, C. cubensis has been found on Miconia spp. native to South America and residing in Melastomataceae. Recent surveys have yielded C. cubensis isolates from new hosts, characterized in this study based on DNA sequences for the ITS and β-tubulin gene regions. These hosts include native Clidemia sericea and Rhynchanthera mexicana (Melastomataceae) in Mexico, and non-native Lagerstroemia indica (Pride of India, Lythraceae) in Cuba. Isolates from these hosts and areas group in the sub-clade of C. cubensis accommodating the South American collections of the fungus. This sub-clade also includes isolates recently collected from Eucalyptus in Cuba, which are used to epitypify C. cubensis. New host records from Southeast Asia include exotic Tibouchina urvilleana from Singapore and Thailand and native Melastoma malabathricum (Melastomataceae) in Sumatra, Indonesia. Consistent with their areas of occurrence isolates from the latter collections group in the Asian sub-clade of C. cubensis. DNA sequence comparisons of isolates from Tibouchina lepidota in Colombia revealed that they represent a new sub-clade within the greater Chrysoporthe clade. Isolates in this clade are described as Chrysoporthe inopina sp. nov., based on distinctive morphological differences.     

Polymorphic Chromosomal Specificity of Centromere Satellite Families in Arabidopsis halleri ssp. gemmifera

The chromosomal localizations of repetitive DNA clusters (ribosomal DNA and centromere satellites) were analyzed by fluorescent in situ hybridization in five strains of Arabidopsis halleri ssp. gemmifera. All five A. gemmifera strains have three chromosome pairs with 45S (5.8S-16S-26S) rDNA loci, and one pair with both 5S and 45S rDNA loci. These localizations are different from that of A. thaliana. Very unusually, there are three families of centromeric satellite DNAs (pAa, pAge1, and pAge2), and they showed polymorphism among the five strains studied. Overall, we found four different centromere satellite compositions. A plant from Fumuro was heterozygous for the chromosome specificities of centromere satellite families, possibly due to a reciprocal translocation involving centromere regions. Changes of centromeric satellite repeats appear to be rapid and frequent events in the history of A. gemmifera, and seem to occur by exchanging clusters as units.     

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 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.     

木奈WRKY同源基因的分离与表达分析

以木奈（Prunus salicina Lindli.var cordata J.Y.Zhang et al.）为试验材料,采用稀释池PCR法,筛选木奈叶片全长均一化cDNA文库,获得了木奈WRKY同源基因的12个全长cDNA分别命名为PsWRKY7、PsWRKY14、PsWRKY20、PsWRKY21、PsWRKY22、PsWRKY29、PsWRKY31、PsWRKY32、PsWRKY33、PsWRKY40、PsWRKY46和PsWRKY75,长度分别为1 517、1 798、2 098、1 177、1 242、1 396、2 135、1 760、2 113、1 047、1 258和700 bp,ORF长度分别为1 095、1 572、1 764、1 068、1 071、1 002、1 944、1 602、1 770、963、1 077和672 bp,分别编码364、523、587、355、357、333、647、533、589、320、358、223个氨基酸。荧光定量结果显示,12个WRKY同源基因在1.0 mmol/L水杨酸处理后,所获得的PsWRKY表达量均显著上调,表明木奈WRKY基因可能是水杨酸诱导防御反应的重要调控因子。     

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.     

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.     

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.     

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 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.     

加州鲈源鰤鱼诺卡氏菌的分离鉴定及致病性

[背景] 鰤鱼诺卡氏菌（Nocardia seriolae）是一种严重危害水产养殖业的病原菌,可引起以体表溃疡、出血及组织器官形成结节为特征的鱼类慢性肉芽肿疾病,目前尚无有效的防治方法。[目的] 明确引起安徽省临泉县某养殖场加州鲈（Micropterus salmonoides）结节病的病原菌,探讨其致病性,为该病的有效防治提供科学依据。[方法] 取肝脏结节病灶接种于TSB培养基分离优势细菌,利用表型检查结合分子生物学方法鉴定分离菌株。进一步通过检测分离菌株的毒力基因、测定其对加州鲈的半数致死量（LD₅₀）以及所感染加州鲈的组织病理学变化与组织载菌量,分析其致病性。[结果] 从病鱼体内分离到一株优势菌株NI,综合NI分离株的表型特性、16S rRNA基因序列与鰤鱼诺卡氏菌参考株相应序列的一致性以及特异性PCR扩增结果,确定其为鰤鱼诺卡氏菌。鰤鱼诺卡氏菌NI分离株携带毒力基因gapA、ibeA和mip,人工回归感染后加州鲈出现与自然病例相似的症状,其对加州鲈的LD₅₀为2.58×10⁶ CFU/尾。组织病理学观察到头肾、心脏、肝脏、胃和脾脏均出现慢性肉芽肿病变,肠管肌层疏松、肠绒毛脱落,肌肉组织中肌纤维疏松、间隙增宽。qPCR检测结果显示,组织中鰤鱼诺卡氏菌载量由高到低依次为头肾、心、肝、胃、脾、肠和肌肉。[结论] 鰤鱼诺卡氏菌是引起此次加州鲈结节病的病原菌,对该菌致病性的研究为加州鲈诺卡氏菌病的防控提供了理论依据。     

乌鳢源杀鱼爱德华菌的分离鉴定及药敏特性研究

【背景】江苏省扬州市某乌鳢养殖场发生疾病,给养殖户造成了严重的经济损失。【目的】确定病因并筛选出敏感药物,为乌鳢相关疾病的防治提供参考。【方法】从患病乌鳢体内分离致病菌,并从形态、生理生化特征、16S rRNA和gyrB基因序列及特异性PCR检测等方面对分离菌株进行鉴定,同时开展人工感染试验分析其致病性,通过纸片扩散法进行药敏特性分析。【结果】从患病乌鳢体内分离获得优势菌株SHL,经形态特征、理化特性、16SrRNA和gyrB基因序列及特异性PCR检测鉴定为杀鱼爱德华菌。进一步人工感染试验证实其对乌鳢有较强的致病性,LD50为1.6×105 CFU/g,发病症状与自然发病症状相似。药敏试验结果显示,该菌株对青霉素、氯霉素、四环素等28种抗菌药物高度敏感,对红霉素中度敏感,对苯唑西啉、克拉霉素、万古霉素等6种药物耐药。【结论】引起江苏省扬州市某养殖场的乌鳢体表溃烂及死亡的病原菌为杀鱼爱德华菌,这是我国首次从淡水鱼类中检出致病性杀鱼爱德华菌,表明该菌的感染谱在扩大,需引起水产养殖领域的重视,在养殖过程中可根据药敏实验结果选用合适的国标渔药进行防治。     

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.     

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.