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.

Utilization of 2-phosphonobutane-1,2,4-tricarboxylic acid as source of phosphorus by environmental bacterial isolates.

Six bacterial strains able to degrade aerobically 2-phosphonobutane-1,2,4-tricarboxylic acid (PBTC) were isolated. The bacteria used PBTC as sole source of phosphorus in the presence of an alternative source of carbon. The microorganisms were taken from various ecosystems, e.g. river water, river sediment and activated sludge. PBTC up to a concentration of 1 mM (270 mg/l) was completely degraded by a defined mixed culture.

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.

cis-chlorobenzene dihydrodiol dehydrogenase (TcbB) from Pseudomonas sp. strain P51, expressed in Escherichia coli DH5alpha(pTCB149), catalyzes enantioselective dehydrogenase reactions.

cis-Chlorobenzene dihydrodiol dehydrogenase (CDD) from Pseudomonas sp. strain P51, cloned into Escherichia coli DH5alpha(pTCB149) was able to oxidize cis-dihydrodihydroxy derivatives (cis-dihydrodiols) of dihydronaphthalene, indene, and four para-substituted toluenes to the corresponding catechols. During the incubation of a nonracemic mixture of cis-1,2-indandiol, only the (+)-cis-(1R,2S) enantiomer was oxidized; the (-)-cis-(S,2R) enantiomer remained unchanged. CDD oxidized both enantiomers of cis-1,2-dihydroxy-1,2,3, 4-tetrahydronaphthalene, but oxidation of the (+)-cis-(1S,2R) enantiomer was delayed until the (-)-cis-(1R,2S) enantiomer was completely depleted. When incubated with nonracemic mixtures of para-substituted cis-toluene dihydrodiols, CDD always oxidized the major enantiomer at a higher rate than the minor enantiomer. When incubated with racemic 1-indanol, CDD enantioselectively transformed the (+)-(1S) enantiomer to 1-indanone. This stereoselective transformation shows that CDD also acted as an alcohol dehydrogenase. Additionally, CDD was able to oxidize (+)-cis-(1R,2S)-dihydroxy-1, 2-dihydronaphthalene, (+)-cis-monochlorobiphenyl dihydrodiols, and (+)-cis-toluene dihydrodiol to the corresponding catechols.

Familial adenomatous polyposis: a submicroscopic deletion at the APC locus in a family with mentally normal patients.

Cytogenetically visible deletions that include the adenomatosis polyposis coli (APC) locus have repeatedly been reported in mentally handicapped polyposis patients. We report on a family with a submicroscopic deletion of about 200 kb including more than the 3' half of the APC gene and the adjacent DP1 gene. The deletion was detected by linkage analysis with flanking and intragenic markers and proven by in situ hybridisation with intragenic cosmid clones. All the familial adenomatous polyposis (FAP) patients and persons at risk in the family show normal behaviour and intelligence. Thus, it is conceivable that at least some of the FAP patients in whom mutations could not be identified by routine methods may have large but submicroscopic deletions.

Valproic acid increases the SMN2 protein level: a well-known drug as a potential therapy for spinal muscular atrophy.

Proximal spinal muscular atrophy (SMA) is a common neuromuscular disorder causing infant death in half of all patients. Homozygous absence of the survival motor neuron gene (SMN1) is the primary cause of SMA, while SMA severity is mainly determined by the number of SMN2 copies. One SMN2 copy produces only about 10% of full-length protein identical to SMN1, whereas the majority of SMN2 transcripts is aberrantly spliced due to a silent mutation within an exonic splicing enhancer in exon 7. However, correct splicing can be restored by over-expression of the SR-like splicing factor Htra2-beta 1. We show that in fibroblast cultures derived from SMA patients treated with therapeutic doses (0.5-500 microM) of valproic acid (VPA), the level of full-length SMN2 mRNA/protein increased 2- to 4-fold. Importantly, this up-regulation of SMN could be most likely attributed to increased levels of Htra2-beta 1 which facilitates the correct splicing of SMN2 RNA as well as to an SMN gene transcription activation. Especially at low VPA concentrations, the restored SMN level depended on the number of SMN2 copies. Moreover, VPA was able to increase SMN protein levels through transcription activation in organotypic hippocampal brain slices from rats. Finally, VPA also increased the expression of further SR proteins, which may have important implications for other disorders affected by alternative splicing. Since VPA is a drug highly successfully used in long-term epilepsy therapy, our findings open the exciting perspective for a first causal therapy of an inherited disease by elevating the SMN2 transcription level and restoring its correct splicing.

Different entities of proximal spinal muscular atrophy within one family.

The molecular analysis of the survival motor neuron (SMN) gene and several closely flanking polymorphic markers in an atypical pedigree with four patients suffering from spinal muscular atrophy (SMA) over two generations has raised new aspects concerning the etiology and the molecular spectrum of autosomal recessive SMA. Three patients in two generations show homozygous deletions of exons 7 and 8 of the telomeric copy of SMN (telSMN), thus confirming the presence of autosomal recessive SMA, with localisation on chromosome 5q12. The fourth SMA patient with mild neurogenic atrophy (confirmed by muscle biopsy and electromyography) shows no homozygous deletion of telSMN but carries a heterozygous deletion of telSMN, as can be deduced from her two affected homozygously deleted children. No intragenic mutation has been identified in the remaining telSMN. In addition, she shares only one SMA chromosome with her affected brother, is haploidentical with two healthy brothers, and has a 31-year-old healthy son, who has inherited an SMN-deleted paternal chromosome and the SMN non-deleted maternal chromosome. These results suggest that this patient either has a neurogenic atrophy of a different origin or exhibits an unusual heterozygous manifestation of SMA 5q12. Interestingly, the two haploidentical telSMN-deleted affected sibs in the second generation show a strikingly discordant clinical picture indicating that, in addition to telSMN mutations, other factors influence the phenotype of SMA in the reported pedigree.

Biotransformation of various substituted aromatic compounds to chiral dihydrodihydroxy derivatives.

The biotransformation of four different classes of aromatic compounds by the Escherichia coli strain DH5alpha(pTCB 144), which contained the chlorobenzene dioxygenase (CDO) from Pseudomonas sp. strain P51, was examined. CDO oxidized biphenyl as well as monochlorobiphenyls to the corresponding cis-2,3-dihydro-2,3-dihydroxy derivatives, whereby oxidation occurred on the unsubstituted ring. No higher substituted biphenyls were oxidized. The absolute configurations of several monosubstituted cis-benzene dihydrodiols formed by CDO were determined. All had an S configuration at the carbon atom in meta position to the substituent on the benzene nucleus. With one exception, the enantiomeric excess of several 1,4-disubstituted cis-benzene dihydrodiols formed by CDO was higher than that of the products formed by two toluene dioxygenases. Naphthalene was oxidized to enantiomerically pure (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. All absolute configurations were identical to those of the products formed by toluene dioxygenases of Pseudomonas putida UV4 and P. putida F39/D. The formation rate of (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene was significantly higher (about 45 to 200%) than those of several monosubstituted cis-benzene dihydrodiols and more than four times higher than the formation rate of cis-benzene dihydrodiol. A new gas chromatographic method was developed to determine the enantiomeric excess of the oxidation products.