A Simple Method for Isolating DNA of High Yield and Quality from Cotton (shape Gossypium hirsutum L.) Leaves

An easy, reproducible and fast procedure to isolate DNA from cotton leaves is described. The addition of 0.5 M glucose in the extraction buffer avoids browning by polyphenolic compounds and improves the quality of DNA for molecular analysis. The DNA yield ranged between 150–400 mg per gram of fresh tissue. The DNA was suitable for digestion by restriction enzymes and amplificatiion by Taq DNA polymerase.     

Vegetation fluctuation in mediterranean dune ponds in relation to rainfall variation and water extraction

Abstract. The structure of dune ponds hygrophytic vegetation has a spatial organisation in belts around the pond basin, closely related to water level and flooding regime. Doñana National Park has an important representation of temporal dune ponds, which are subjected to rainfall fluctuations and may be suffering the impact of water extraction from the neighbouring tourist resort. Permanent transects in a vegetation complex of five dune ponds have been monitored during a eight year period (1990-1997). This period was characterised by a number of dry years (annual rainfall around 300 mm), located between two wet cycles (800-900 mm). Transects were analysed in 1990 (wet period), 1994 (dry) and 1997 (wet) by hierarchical agglomera-tive clustering. During the dry period hygrophytic species showed regression, with a high mortality of some species like Ulex minor, while the xerophytic species advanced to lower areas. Seedlings of some xerophytic species colonised the dry surface of the pond basin. The lowering of the water table varied in the different ponds, ranging from 312 to 190 cm depending on topography and the distance to the pumping area. The new period of flooding during 1995-96 and 1996-97 cycles provided the opportunity for hygrophytic spe cies to re-establish themselves in their original places. This study suggest that changes in vegetation are caused by the interaction between weather conditions and human disturbance (water extractions). In our example man-made disturbance is more marked during the dry periods while wet periods tend to obscure the effects of water extractions. From a management perspective, long-term monitoring of water table and vegetation structure is revealed as a key procedure to the management of land-water ecotones on pressured areas and threatened habitats.     

Sequence heterogeneity and anomalous electrophoretic mobility of kinetoplast minicircle DNA from Leishmania tarentolae

Several unit-length minicircles from the kinetoplast DNA of Leishmania tarentolae were cloned into pBR322 and into M13 phage vectors. The complete nucleotide sequences of three different partially homologous minicircles were obtained. The molecules contained a region of approx. 80% sequence homology extending for 160–270 bp and a region unique to each minicircle. A 14-mer was found to be conserved in all kinetoplast minicircle sequences reported to date. The frequency distributions of various minicircle sequence classes in L. tarentolae were obtained by quantitative gel electrophoresis and by examination of the “T ladder” patterns of minicircles randomly cloned into M13 at several sites. By these methods we could assign approx. 50% of the total minicircle DNA into a minimum of five sequence classes. A sequence-dependent polyacrylamide gel migration abnormality was observed with several minicircle fragments both cloned and uncloned. The abnormality was dependent on the presence of a portion of the conserved region of the minicircle.     

Measurement of 7-dehydrocholesterol and cholesterol in hair can be used in the diagnosis of Smith-Lemli-Opitz syndrome

7-dehydrocholesterol (7-DHC) and cholesterol (CHOL) are biomarkers of Smith-Lemli-Opitz Syndrome (SLOS), a congenital autosomal recessive disorder characterized by elevated 7-DHC level in patients. Hair samples have been shown to have great diagnostic and research value, which has long been neglected in the SLOS field. In this study, we sought to investigate the feasibility of using hair for SLOS diagnosis. In the presence of antioxidants (2,6-ditert-butyl-4-methylphenol and triphenylphosphine), hair samples were completely pulverized and extracted by micro-pulverized extraction in alkaline solution or in n-hexane. After microwave-assisted derivatization with N,O-Bis(trimethylsilyl)trifluoroacetamide, the analytes were measured by GC-MS. We found that the limits of determination for 7-DHC and CHOL were 10 ng/mg and 8 ng/mg, respectively. In addition, good linearity was obtained in the range of 50–4000 ng/mg and 30–6000 ng/mg for 7-DHC and CHOL, respectively, which fully meets the requirement for SLOS diagnosis and related research. Finally, by applying the proposed method to real hair samples collected from 14 healthy infants and two suspected SLOS patients, we confirmed the feasibility of hair analysis as a diagnostic tool for SLOS. In conclusion, we present an optimized and validated analytical method for the simultaneous determination of two SLOS biomarkers using human hair.     

Sequences of cDNAs for human salivary and pancreatic α-amylases

The nucleotide sequences of the cloned human salivary and pancreatic α-amylase cDNAs correspond to the continuous mRNA sequences of 1768 and 1566 nucleotides, respectively. These include all of the amino acid coding regions. Salivary cDNA contains 200 bp in the 5′-noncoding region and 32 in the 3′-noncoding region. Pancreatic cDNA contains 3 and 27 bp of 5′- and 3′-noncoding regions, respectively. The nucleotide sequence humology of the two cDNAs is 96% in the coding region, and the predicted amino acid sequences are 94% homologous.Comparison of the sequences of human α-amylase cDNAs with those previously obtained for mouse α-amylase genes (Hagenbuchle et al., 1980; Schibler et al., 1982) showed the possibility of gene conversion between the two genes of human α-amylase.     

Simple and efficient cell disruption of extremely small quantities of mycelium of phytopathogenic mycotoxin-producing moulds for quantitative extraction of genomic DNA

DNA was efficiently and quantitatively isolated from extremely small quantities of mycelia (0.1–10 mg) of different phytopathogenic moulds by grinding freeze-dried mycelia with glass beads and then using a commercial DNA extraction kit. The efficiency of disruption of the mycelia and the quantitative DNA extraction was proved by microscopy and the quantification of isolated DNA by real time PCR. Presented at the 27^th Mykotoxin-Workshop, Dortmund, Germany, June 13–15, 2005 Financial support: German Research Foundation (DFG grant Pr 708/2). J.M. thanks the Cusanuswerk for a doctoral scholarship     

The effects of Bombyx mori silk strain and extraction time on the molecular and biological characteristics of sericin

Sericin was extracted from three strains of Thai Bombyx mori silk cocoons (white shell Chul1/1, greenish shell Chul3/2, and yellow shell Chul4/2) by a high-pressure and high-temperature technique. The characteristics of sericin extracted from different fractions (15, 45, and 60 min extraction process) were compared. No differences in amino acid composition were observed among the three fractions. For all silk strains, sericin extracted from a 15-min process presented the highest molecular weight. The biological potential of the different sericin samples as a bioadditive for 3T3 fibroblast cells was assessed. When comparing sericin extracted from three silk strains, sericin fractions extracted from Chul4/2 improved cell proliferation, while sericin from Chul 1/1 activated Type I collagen production to the highest extent. This study allows the natural variability of sericin obtained from different sources and extraction conditions to be addressed and provides clues for the selection of sericin sources.     

Horizontal transfer of a mitochondrial plasmid

Direct evidence for horizontal transfer of a mitochodnrial plasmid from the discomyceteAscobolus immersus to the pyrenomycetePodospora anserina is presented. Southern blot hybridisation analysis, polymerase chain reaction (PCR) amplification, and DNA sequencing demonstrate transmission of a linear plasmid upon hyphal contact. DNA extraction from isolated organelles indicates a mitochondrial localisation for the plasmid inP. anserina. This is the first report of horizontal gene transfer among unrelated fungi. These results have important evolutionary implications for plasmid propagation in fungi.     

Plasmid-coded degradation of ethylbenzene and 1-phenylethanol in Pseudomonas fluorescens

Abstract Pseudomonas fluorescens EB carries genes for the catabolism of ethylbenzene and 1-phenylethanol on a plasmid. The size of the plasmid as measured by analysis of agarose electrophoresis gels after restriction endonuclease hydrolysis, was 253–267 kb. By treatment with Mitomycin C, mutants of EB strain were obtained bearing a plasmid which had undergone an extensive deletion of about 80 kb. These mutants have lost the ability to grow on ethylbenzene and 1-phenylethanol as well as to synthesize meta-cleavage enzymes.     

Anwendung der automatisierten Festphasenextraktion bei der Bestimmung von Ochratoxin A

For some foodstuffs, determination of the mycotoxin ochratoxin A (OTA) requires time consuming clean up by means of solid phase extraction (SPE). Therefore a system for automated SPE was tested for cleaning up roasted coffee as a possible way of shortening preparation time. Validation of the method in accordance to the so called “Concept '98” led to a LOD of 0.2 μg/kg and a recovery rate of 92%. By using the described procedure with samples of roasted coffee the OTA contents varied between the LOD and 3.4 μg/kg. This method was also used to determine ochratoxin A in liquorice roots, ginger and valerian.

Presented at the 26^th Mykotoxin Workshop in Herrsching, Germany, May 17–19, 2004