Comparison of different methods for the recovery of DNA from spores of mycotoxin-producing moulds in spiked food samples.

Several food samples were spiked with fungal conidia to test the efficiency of different cell disruption methods and DNA extraction kits for subsequent molecular detection. For disrupting the firm cell walls of the spores, two different pretreatment methods, namely sonication and bead beating, were tested against no pretreatment. The subsequent DNA extraction and purification was performed using three different DNA extraction methods, which are based on a diverse combination of extraction principles, such as precipitation, thermic-enzymatic lysis, pH-enhancement and bonding with a silica membrane. The aim of the study was to find out the suitable pretreatment and DNA extraction method for the recovery of detectable amounts of fungal DNA from different food matrices. Significance and impact of the study: The choice of 'ready-to-use' commercial kits and methods has been of great importance regarding the recovery of extracted DNA. However, these commercially available kits are neither effective nor time-efficient when extracting DNA from fungal spores embedded in complex food matrices. Different extraction principles were compared and their effectiveness tested using real-time PCR. The combination of different principles for the extraction and purification of DNA was found as the most efficient method (quantity and purity) to obtain DNA from moulds and their spores from food samples.

Development of deoxynivalenol contents in relation to the PCR detection of potentially trichothecene producingFusarium spp. during storage of wheat.

Development of deoxynivalenol (DON) in wheat with a low contamination withFusarium spp. was investigated under suboptimal storage conditions (17% and 20% grain moisture, 20°C). The influence of storage on the relative DNA content of potential DON producers was also determined. The DON contents were quantified using an ELISA. The Tox5 PCR was used for the detection of potential trichothecene producers and for the estimation of their relative DNA content. ThegaoA gene was subsequently amplified by PCR to detect specificallyFusarium graminearum. The concentration ofF. graminearum DNA was semiquantitatively determined using a Light Cycler™. The DON concentrations increased during storage trials but the intensity of PCR signals decreased.

De novo rearrangements found in 2% of index patients with spinal muscular atrophy: mutational mechanisms, parental origin, mutation rate, and implications for genetic counseling.

Spinal muscular atrophy (SMA) is a relatively common autosomal recessive neuromuscular disorder. We have identified de novo rearrangements in 7 (approximately 2%) index patients from 340 informative SMA families. In each, the rearrangements resulted in the absence of the telomeric copy of the survival motor neuron (SMN) gene (telSMN), in two cases accompanied by the loss of the neuronal apoptosis-inhibitory protein gene . Haplotype analysis revealed unequal recombination in four cases, with loss of markers Ag1-CA and C212, which are near the 5' ends of the SMN genes. In one case, an interchromosomal rearrangement involving both the SMN genes and a regrouping of Ag1-CA and C212 alleles must have occurred, suggesting either interchromosomal gene conversion or double recombination. In two cases, no such rearrangement was observed, but loss of telSMN plus Ag1-CA and C212 alleles in one case suggested intrachromosomal deletion or gene conversion. In six of the seven cases, the de novo rearrangement had occurred during paternal meiosis. Direct detection of de novo SMA mutations by molecular genetic means has allowed us to estimate for the first time the mutation rate for a recessive disorder in humans. The sex-averaged rate of 1.1 x 10(-4), arrived at in a proband-based approach, compares well with the rate of 0.9 x 10(-4) expected under a mutation-selection equilibrium for SMA. These findings have important implications for genetic counseling and prenatal diagnosis in that they emphasize the relevance of indirect genotype analysis in combination with direct SMN-gene deletion testing in SMA families.

Missense mutations in exon 6 of the survival motor neuron gene in patients with spinal muscular atrophy (SMA).

Spinal muscular atrophy (SMA) is a frequent autosomal recessive neurodegenerative disorder leading to weakness and atrophy of voluntary muscles. The survival motor neuron gene (SMN) is a strong candidate for SMA and present in two highly homologous copies (telSMN and cenSMN) within the SMA region (5q11.2-q13.3). More than 90% of SMA patients show homozygous deletions of at least exon 7 of telSMN, whereas absence of cenSMN seems to have no clinical consequences. In 23 non-deleted SMA patients, we searched for intragenic mutations of the SMN genes in exons 1-7 and the promotor region by single strand conformation analysis. We identified two different missense mutations, S2621 and T2741, in exon 6 of telSMN in three independent SMA families, providing further evidence for the telSMN gene as a SMA determining gene. Both mutations, as well as two previously described mutations (Y272C and G279V) are located within a highly conserved interval from codon 258 to codon 279 which seems to be an important functional domain of the telSMN protein. Recently, this region has been shown to contain a tyrosine/glycine-rich motif, which is also present in various RNA binding proteins, suggesting a potential role of SMN in RNA metabolism. Missense mutations might be useful for in vivo and transgenic experiments and further investigations on understanding the function of the telSMN protein.

Hybrid survival motor neuron genes in patients with autosomal recessive spinal muscular atrophy: new insights into molecular mechanisms responsible for the disease.

Spinal muscular atrophy (SMA) is a frequent autosomal recessive neurodegenerative disorder leading to weakness and atrophy of voluntary muscles. The survival motor-neuron gene (SMN), a strong candidate for SMA, is present in two highly homologous copies (telSMN and cenSMN) within the SMA region. Only five nucleotide differences within the region between intron 6 and exon 8 distinguish these homologues. Independent of the severity of the disease, 90%-98% of all SMA patients carry homozygous deletions in telSMN, affecting either exon 7 or both exons 7 and 8. We present the molecular analysis of 42 SMA patients who carry homozygous deletions of telSMN exon 7 but not of exon 8. The question arises whether in these cases the telSMN is truncated upstream of exon 8 or whether hybrid SMN genes exist that are composed of centromeric and telomeric sequences. By a simple PCR-based assay we demonstrate that in each case the remaining telSMN exon 8 is part of a hybrid SMN gene. Sequencing of cloned hybrid SMN genes from seven patients, as well as direct sequencing and single-strand conformation analysis of all patients, revealed the same composition in all but two patients: the base-pair differences in introns 6 and 7 and exon 7 are of centromeric origin whereas exon 8 is of telomeric origin. Nonetheless, haplotype analysis with polymorphic multicopy markers, Ag1-CA and C212, localized at the 5' end of the SMN genes suggests different mechanisms of occurrence, unequal rearrangements, and gene conversion involving both copies of the SMN genes. In approximately half of all patients, we identified a consensus haplotype, suggesting a common origin. Interestingly, we identified a putative recombination hot spot represented by recombination-stimulating elements (TGGGG and TGAGGT) in exon 8 that is homologous to the human deletion-hot spot consensus sequence in the immunoglobulin switch region, the alpha-globin cluster, and the polymerase alpha arrest sites. This may explain why independent hybrid SMN genes show identical sequences.

Molecular analysis of candidate genes on chromosome 5q13 in autosomal recessive spinal muscular atrophy: evidence of homozygous deletions of the SMN gene in unaffected individuals.

Proximal spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder characterized by degeneration of anterior horn cells in the spinal cord leading to weakness and wasting of voluntary muscles. Here we present the molecular analysis of both SMA candidate genes, the survival motor neuron gene (SMN; exons 7 and 8) and the neuronal apoptosis inhibitory protein gene (NAIP; exons 5, 6 and 13), in 195 patients and 348 parents of SMA families mainly of German origin. The SMN gene is homozygously deleted for both exons 7 and 8 or exon 7 only in 96% of type I SMA, 94% of type II SMA and 82% of type III SMA as well as in 0.3% of SMA parents. The NAIP gene is homozygously deleted in 46% of type I SMA, 17% of type II SMA, 7% of type III SMA and 2% of SMA parents. The frequencies of deletions in patients for both genes, SMN and NAIP, correspond to those for the NAIP gene only. SMA patients of this series who did not show deletions were clinically indistinguishable from deleted patients. In addition to one unaffected mother of a type II SMA patient, we found homozygous deletions of the SMN gene exons 7 and 8 in six further unaffected individuals, all sibs of type II and III patients. These belonged to four families with affected and unaffected sibs who showed identical haplotypes for all SMA flanking markers; therefore, we had regarded these families as chromosome 5 unlinked. All seven unaffected individuals in whom we detected SMA deletions do not show any signs of muscle weakness and are physically inconspicuous. The largest divergence between age at onset of an affected subject and the present age of unaffected deleted sibs is four decades now. The occurrence of SMN deletions in unaffected individuals suggests that other genes or mechanisms may be necessary to produce the SMA phenotype.

Mapping of the spinal muscular atrophy (SMA) gene to a 750-kb interval flanked by two new microsatellites.

The gene for autosomal recessive proximal spinal muscular atrophy (SMA) has recently been mapped between D5S629 and D5S557. We report here a new single-locus microsatellite A31 (D5S823) and two multicopy microsatellites 97T-CA and 95/23-CA. The marker A31 maps to the region of overlap between YACs y116, y55 and y122, distal to D5S629; 97T-CA originates from a cosmid corresponding to the STS 97T, localized distally to A31, while 95/23-CA derives from a cosmid corresponding to the STS 97U, localized proximally to D5S557. We tested all our key recombinant families with these markers. In one type I/II SMA family, a recombinant was found that placed the SMA locus distal to D5S823. Homozygosity mapping in a consanguineous type I SMA family indicates that the SMA gene lies proximal to 95/23-CA. Thus, the two new markers, A31 and 95/23-CA further refine the SMA gene to an approximately 750-kb interval.

Large linkage analysis in 100 families with autosomal recessive spinal muscular atrophy (SMA) and 11 CEPH families using 15 polymorphic loci in the region 5q11.2-q13.3.

The autosomal recessive proximal spinal muscular atrophy (SMA) gene was mapped to the region 5q11.2-q13.3 in 1990. Here, we present a large genetic linkage study of 100 SMA families and 11 CEPH families using 14 polymorphic simple sequence repeats (SSRs) and one RFLP in the region 5q11.2-q13.3. The genetic interval between the closest SMA flanking loci D5S435 and D5S557 comprises 1 cM at zmax = 27.94. Two recombinants were identified between the SMA gene and the closest telomeric marker D5S557 (theta = 0.02 at zmax = 8.63). The first places the SMA gene centromeric to this marker; the second suggests a double recombinant at D5S557, which is very unlikely. More likely explanations are discussed in the paper. No recombinant was found between D5S435 and the SMA gene (theta = 0.00 at zmax = 25.36). We localized a recently described polymorphic marker, D5S351 (Hudson et al., 1992), close to the SMA (theta = 0.00 at zmax = 19.01) and the 3'MAP1B gene (theta = 0.01 at zmax = 38.76). Due to its high PIC value of 0.70, it represents a very useful marker for prenatal diagnosis. In addition, we developed a new reverse primer for the nearest centromeric locus D5S435 (Soares et al., 1993), a useful marker for prenatal diagnosis, which has been very difficult to amplify in the past. Three of the markers presented here are newly developed polymorphic SSRs (one tetranucleotide repeat, D5S507/W15CATT, and two dinucleotide repeats, D5S544/C88.2GT and D5S682/C88.3GT). These markers are too far from the SMA gene to be relevant for cloning; nevertheless, as part of the human genome project, they are contributing to the fine genetic mapping of the region 5q11.2-q13.3. The most likely order of the loci based on two-point and multipoint linkage analyses as well as on specific recombination events and physical mapping studies is D5S76-D5S507- D5S6-D5S125-D5S680-D5S435-SMA-D5S557- D5S351-5'MAP1B-3'MAP1B-JK53CA1/2-(D5S127- D5S39)-(D5S544-D5S682). In general, the genetic distances obtained from the SMA and CEPH families are comparable.

Efficiency of various strategies and materials to generate new markers: saturating the region 5q11.2-q13.3 with 30 new randomly distributed clones.

Several different strategies and materials were used for saturating the region 5q11.2-q13.3 with new, randomly distributed markers: isolation of human clones from three chromosome-5-specific libraries (a BssHII endclone phage library from the somatic cell hybrid H64 and two total genomic phage libraries from radiation hybrids IH12 and IH132), as well as Alu-PCR from chromosome-5-specific radiation hybrids with overlapping fragments in the region around the spinal muscular atrophy locus, followed either by direct isolation of Alu-PCR products or hybridization of Alu-PCR products to chromosome-5-gridded cosmid libraries. 253 human phage and cosmid clones were mapped to various parts of chromosome 5 by deletion mapping to somatic cell hybrid panels. 30 of these clones were mapped into the region 5q11.2-q13.3, 9 of which are flanking rate cutting BssHII-sites, known to be, often, starting points for genes. They represent excellent starting material for the development of new polymorphic markers and sequence-tagged sites, for YAC screening and building of contigs, as well as for direct isolation of genes.

Molecular cloning and nucleotide sequence of a new plasmid-coded Klebsiella pneumoniae beta-lactamase gene (SHV-2a) responsible for high-level cefotaxime resistance.

Patient specimen isolates of Klebsiella pneumoniae exhibiting an MIC of greater than 128 mg/l for cefotaxime were shown to produce a beta-lactamase with a pI of 7.6, which is encoded on a 66 kb conjugative plasmid. A 3.5 kb Bam HI fragment of this plasmid was cloned into pLG339 and totally sequenced. The nucleotide sequence of the beta-lactamase gene presented 99% homology to those of SHV-2 and SHV-3, the deduced amino acid sequence differed from both enzymes in one and two positions, respectively, leading to the denomination SHV-2a for the new enzyme. Since the kinetic data of SHV-2a and SHV-2 are similar, too, quantitative effects mediated by distinctly different promotor regions are thought to be responsible for the elevated MIC for cefotaxime induced by SHV-2a.

Different promoters of SHV-2 and SHV-2a beta-lactamase lead to diverse levels of cefotaxime resistance in their bacterial producers.

Clinical Klebsiella pneumoniae isolates as well as Escherichia coli transformants producing the beta-lactamases SHV-2 or SHV-2a demonstrate MIC values for cefotaxime of 4 mg l-1 or 64 to greater than 128 mg l-1, respectively. The beta-lactamases differ by one possibly insignificant amino acid exchange at position number 10 of the mature protein; their kinetic parameters are rather similar. The 5' untranslated regions of both corresponding genes show no homology starting 74 nucleotides upstream to the start codon. Hybridization of intragenically annealing oligonucleotides to dot-blotted serial dilutions of total cellular RNA from E. coli transformants harbouring these genes cloned into the same vector plasmid gave a positive signal down to 1.2 micrograms (SHV-2) and 0.32 to 0.16 micrograms (SHV-2a), indicating a four to eight times higher amount of specific transcript in the case of SHV-2a. By primer extension analysis and S1 nuclease digestion the starting point to transcription was located 100 nucleotides (SHV-2) and 50 nucleotides (SHV-2a) in front of the start codon. No other transcripts of different length could be detected after prolonged exposure. Northern blot analysis demonstrated the length of the beta-lactamase mRNA to be about 1.6 kb in both cases, thus comprising a potential open reading frame downstream of the two enzymes' genes. Selective PCR amplification of both promoter regions and of the structural gene of SHV-2 and subsequent combined cloning of each of the promoters and the SHV-2 gene into pBGS19 using a BamHI restriction site introduced by three point mutations into the cloned sequences was employed to transforms E. coli DH5 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular characterization of a new plasmid-encoded SHV-type beta-lactamase (SHV-2 variant) conferring high-level cefotaxime resistance upon Klebsiella pneumoniae.

Between 1986 and 1988, multiresistant Klebsiella pneumoniae strains exhibiting high-level cefotaxime resistance were isolated from patient specimens particularly of the intensive care units of the Aachen Technical University Hospital. The resistance gene responsible was shown to be encoded on a conjugative 66 kb plasmid designated pZMP1. The MIC values for cefotaxime of the original isolates and the transconjugants were greater than 128 mg l-1 and 64 mg l-1, respectively. Isoelectric focusing of protein preparations from the transconjugants showed a beta-lactamase with a pI of 7.6. A 3.6 kb BamHI fragment containing the beta-lactamase gene was cloned into pLG339 resulting in the recombinant plasmid pZMP1-1. A restriction map of the cloned insert was established and PstI subfragments of the insert were further subcloned into pBGS18. The nucleotide sequence of the complete 3.6 kb fragment was determined. Within 3663 bp an open reading frame of 858 kb was found to show 99% homology to the SHV-2 and -3 nucleotide sequences. The deduced amino acid sequence differed in one and two positions, respectively, from these established SHV enzymes. The 3' noncoding sequence exhibited nearly perfect homology to that of SHV-2, but the 5' upstream sequence showed homology of less than 50% to the corresponding SHV-2 sequence, indicating an altered promoter region of the variant SHV-enzyme. Kinetic analysis of the beta-lactamase revealed a 50-100% elevated hydrolytic effectivity on cefotaxime in comparison to other SHV enzymes. Cefoxitin, ceftazidime, aztreonam and imipenem were not hydrolysed by the enzyme. The variant enzyme was inhibited by commonly available beta-lactamase inhibitors. Clavulanic acid had the highest affinity for the enzyme and the greatest effectivity in blocking its action. Based on the genetic and kinetic data we propose to classify the enzyme as a new variant beta-lactamase of the SHV-type and name it SHV-2a.