Array-comparative genomic hybridization analysis in patients with Müllerian fusion anomalies.

Fusion anomalies of the Müllerian ducts are associated with an increased risk for miscarriage and premature labor. In most cases polygenic-multifactorial inheritance can be assumed but autosomal-dominant inheritance with reduced penetrance and variable manifestation should be considered. We performed array-comparative genomic hybridization (CGH) analysis in a cohort of 103 patients with Müllerian fusion anomalies. In 8 patients we detected microdeletions and microduplications in chromosomal regions 17q12, 22q11.21, 9q33.1, 3q26.11 and 7q31.1. The rearrangement in 17q12 including LHX1 and HNF1ß as well as in 22q11.21 have already been observed in MRKHS (Mayer-Rokitansky-Küster-Hauser syndrome). In summary, we (1) detected causative micro-rearrangements in patients with Müllerian fusion anomalies, (2) show that Müllerian fusion anomalies and MRKHS may have a common etiology, and (3) identified new candidate genes for Müllerian fusion anomalies.

Mutations in WNT9B are associated with Mayer-Rokitansky-Küster-Hauser syndrome.

Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is a well-known malformation pattern of the Müllerian ducts (MDs) characterized by congenital absence of the uterus and vagina. To date, most cases remain unexplained at molecular level. As female Wnt9b-/- mice show a MRKHS-like phenotype, WNT9B has emerged as a promising candidate gene for this disease. We performed retrospective sequence analyses of WNT9B in 226 female patients with disorders of the MDs, including 109 patients with MRKHS, as well as in 135 controls. One nonsense mutation and five likely pathogenic missense mutations were detected in WNT9B. Five of these mutations were found in cases with MRKHS accounting for 4.6% of the patients with this phenotype. No pathogenic mutations were detected in the control group (p = 0.017). Interestingly, all of the MRKHS patients with a WNT9B mutation were classified as MRKHS type 1, representing 8.5% of the cases from this subgroup. In previous studies, two of the patients with a WNT9B mutation were found to carry either an additional deletion of LHX1 or a missense mutation in TBX6. We conclude that mutations in WNT9B were frequently associated with MRKHS in our cohort and some cases may be explained by a digenic disease model.

Ectopic expression of EFFECTOR OF TRANSCRIPTION perturbs gibberellin-mediated plant developmental processes.

The plant hormone gibberellin (GA) is known to modulate various aspects of plant cell differentiation and development. The current model of GA-mediated regulation is based on a de-repressible system and includes specific protein modification and degradation. HRT, a zinc finger protein from barley has been shown to have GA-dependent transcriptional repressing activity on the seed-specific alpha-amylase promoter [Raventos, D., Skriver, K., Schlein, M., Karnahl, K., Rogers, S.W., Rogers, J.C. and Mundy, J. 1998. J. Biol. Chem. 273: 23313-23320]. Here we report the characterization of a dicot homologue from Brassica napus (BnET) and provide evidence for its role in GA response modulation suggesting that this could be a conserved feature of this gene family. When BnET is ectopically expressed in either Arabidopsis or tobacco the phenotypes include dwarfism due to shorter internodes and late flowering, reduced germination rate, increased anthocyanin content and reduced xylem lignification as a marker for terminal cell differentiation. Transient expression in protoplasts supports the notion that this most likely is due to a transcriptional repression of GA controlled genes. Finally, histological analysis showed that in contrast to other GA deficient mutants the shorter internodes were due to fewer but not smaller cells, suggesting a function of BnET in GA-mediated cell division control.

Amino acid permeases in developing seeds of Vicia faba L.: expression precedes storage protein synthesis and is regulated by amino acid supply.

Full length cDNAs encoding three amino acid permeases were isolated from seed-specific libraries of Vicia faba. The predicted proteins VfAAP1, VfAAP3 and VfAAP4 share up to 66% identity among themselves. Functional characterization of VfAAP1 and VfAAP3 in a yeast mutant showed that these permeases transport a broad range of amino acids. However, VfAAP1 had a preference for cysteine and VfAAP3 for lysine and arginine. VfAAP1 was highly expressed in cotyledons at early developmental stages and moderately in other sink tissues. Its peak of expression in cotyledons corresponded to the appearance of storage protein transcripts, suggesting that this transporter fulfills an important role in providing amino acids for storage protein biosynthesis. VfAAP3 was expressed most abundantly in maternal tissues, that is in roots, stems, gynoecia, pods and seed coats at different developmental stages. VfAAP4 transcripts could not be detected by northern hybridization. In situ hybridization showed that VfAAP1 mRNA is distributed throughout cotyledon storage parenchyma cells, but could not be detected in the abaxial epidermal cell layer. It also accumulate in the chlorenchyma and thin-walled parenchyma cells of seed coats. VfAAP1 mRNA levels were lower in cotyledons cultured in the presence of glutamine, whereas expression of a vicilin storage protein gene was up-regulated under similar conditions. Cysteine repressed the expression of the GUS reporter gene under control of the VfAAP1 promoter, suggesting that this transporter is modulated at the transcriptional level. Regulation of amino acid transport in relation to storage protein accumulation is discussed.

Molecular characterization of cDNAs encoding G protein alpha and beta subunits and study of their temporal and spatial expression patterns in Nicotiana plumbaginifolia Viv.

We have isolated cDNA sequences encoding alpha and beta subunits of potential G proteins from a cDNA library prepared from somatic embryos of Nicotiana plumbaginifolia Viv. at early developmental stages. The predicted NPGPA1 and NPGPB1 gene products are 75-98% identical to the known respective plant alpha and beta subunits. Southern hybridizations indicate that NPGPA1 is probably a single-copy gene, whereas at least two copies of NPGPB1 exist in the N. plumbaginifolia genome. Northern analyses reveal that both NPGPA1 and NPGPB1 mRNA are expressed in all embryogenic stages and plant tissues examined and their expression is obviously regulated by the plant hormone auxin. Immunohistological localization of NPGPalpha1 and NPGPbeta1 preferentially on plasma and endoplasmic reticulum membranes and their immunochemical detection exclusively in microsomal cell fractions implicate membrane association of both proteins. The temporal and spatial expression patterns of NPGPA1 and NPGPB1 show conformity as well as differences. This could account for not only cooperative, but also individual activities of both subunits during embryogenesis and plant development.

Gene regulation during late embryogenesis: the RY motif of maturation-specific gene promoters is a direct target of the FUS3 gene product.

The Arabidopsis mutants fus3 and abi3 show pleiotropic effects during embryogenesis including reduced levels of transcripts encoding embryo-specific seed proteins. To investigate the interaction between the B3-domain-containing transcription factors FUS3 and ABI3 with the RY cis-motif, conserved in many seed-specific promoters, a promoter analysis as well as band-shift experiments were performed. The analysis of promoter mutants revealed the structural requirements for the function of the RY cis-element. It is shown that both the nucleotide sequence and the alternation of purin and pyrimidin nucleotides (RY character) are essential for the activity of the motif. Further, it was shown that FUS3 and ABI3 can act independently of each other in controlling promoter activity and that the RY cis-motif is a target for both transcription factors. For FUS3, which is so far the smallest known member of the B3-domain family, a physical interaction with the RY motif was established. The functional and biochemical data demonstrate that the regulators FUS3 and ABI3 are essential components of a regulatory network acting in concert through the RY-promoter element to control gene expression during late embryogenesis and seed development.

Calreticulin expression in plant cells: developmental regulation, tissue specificity and intracellular distribution.

The tissue-specific expression pattern and the intracellular distribution of the Ca(2+)-binding protein calreticulin at the mRNA and protein levels have been studied during somatic and zygotic embryogenesis of Nicotiana plumbaginifolia Viv. A full-length cDNA sequence encoding calreticulin was isolated from a lembda Zap cDNA library from early developmental stages of somatic embryogenesis. The deduced amino acid sequence of the calreticulin from N. plumbaginifolia shows high homology to the corresponding proteins of tobacco (98.2% identity), maize (80%) and barley (76.5%), and more than 55% homology to animal calreticulins, and the sequence motifs with established functions found in calreticulins of other species were quite conserved. Northern experiments revealed a developmental regulation of the calreticulin transcript with a maximum during the early stages of somatic embryogenesis and an auxin dependence during in-vitro cell culture. alpha-Naphthaleneacetic acid stimulated calreticulin expression whereas 2,4-dichlorophenoxyacetic acid reduced it. Immunohistological analysis of calreticulin distribution in the ovaries during zygotic embryogenesis showed that calreticulin biosynthesis started tissue specifically, with a high abundance in the endothelium of the integument in the ovules, followed by calreticulin accumulation in the embryo proper and in the associated endosperm at the late globular stage of embryogenesis. Using immunogold labeling, calreticulin was intracellularly localized with a high abundance to the Golgi compartment and to patches on the surface of dividing protoplasts. Smaller amounts were found in the endoplasmic reticulum and plasma membranes. The functional role of calreticulin in posttranslational processing and translocation processes, apart from its postulated function in cellular Ca2+ homeostasis, is discussed.

Long-term cultures of barley synthesize and correctly deposit seed storage proteins.

Long-term cultures of four different cultivars of barley (Hordeum vulgare L.) have been established. Both callus and suspension cultures formed embryogenic structures at high frequency even after more than 18 months of culture. These compact proembryogenic cell clusters synthesize seed storage globulins whereas loose cell aggregates in callus culture and suspension cultures of fine dispersed consistency were free of globulins. Globulin synthesis was especially intense in compact structures of callus cultures established from suspension culture-derived protoplasts. Within the cells storage globulins are deposited in the vacuolar compartment as in zygotic embryos. The molecular data provided recommend the system for studies on factors determining seed protein gene expression and intracellular protein transport.

Temporal relationship between force, ATPase activity, and myosin phosphorylation during a contraction/relaxation cycle in a skinned smooth muscle.

The temporal relationship between myosin phosphorylation, contractile force and ATPase activity was studied in skinned preparations from the guinea-pig Taenia coli. When free Calcium concentration [( Ca2+]) was increased from pCa (-log[Ca2+]) 9 to pCa 4.5 at low calmodulin concentration (0.05 microM), ATPase activity and myosin light-chain phosphorylation rose quickly, while the increase in force and stiffness was delayed. The time-course of tension increase was faster at higher calmodulin concentrations (5 microM), although the maximal level of phosphorylation was unchanged. Lowering the calcium concentration from pCa 4.5 to pCa 9 at the plateau of contraction caused a rapid decrease in ATPase activity and in myosin phosphorylation, while force and stiffness decayed more slowly. The force decay could be accelerated by inorganic phosphate. These results suggest that, during contraction, force may be produced actively by phosphorylated and ATP-splitting crossbridges, but may be maintained by dephosphorylated crossbridges which cycle slowly. However, force could also be modulated by calmodulin and inorganic phosphate in a manner not involving an alteration in the extent of myosin phosphorylation.

Comparative Investigations on the Metabolism of 2-(2,4-Dichlorophenoxy)Isobutyric Acid in Plants and Cell Suspension Cultures of Lycopersicon esculentum.

The metabolism of [(14)CH(3)]2-(2,4-dichlorophenoxy)isobutyric acid (DIB) was studied in plants and cell suspension cultures of Lycopersicon esculentum Mill. sp. ;Lukullus'. Both plants and cells in suspension culture showed a rapid uptake of DIB from nutrient media. The metabolites, isolated by extraction with methanol and separated by chromatographic methods, were identified by enzymic, chemical, and spectrometric methods. Two conjugates of the carboxyl with 2 and 3 moles glucose per mole DIB and, to a smaller extent, its beta-d-glucopyranosyl ester, were formed in both intact plants and cell suspension cultures, but there were quantitative differences.

Evidence that a Single Polypeptide Catalyses the Two Step Conversion of Orotate to UMP in Cells from a Tomato Suspension Culture.

A single polypeptide catalysing the two step conversion of orotate to UMP in cultured tomato cells was purified to near homogeneity as judged by analytical disc gel electrophoresis. After electrophoresis of the dodecyl sulphate denaturated enzyme one single protein band appeared from which both enzyme activities could be renaturated by addition of Triton X-100. As introduced by McClard et al. (Biochemistry 19, 4699-4706, 1980) for the mammalian system, this plant enzyme should be termed UMP synthase, consequently. The enzyme consists of a single polypeptide chain of a molecular weight of about 51,000. Molecular weight determination by gel filtration under non-denaturating conditions gave a value of about 100,000, suggesting dimer formation in vivo. The enzyme contains thiol groups essential for enzyme activity. Kinetic characteristics of the orotate phosphoribosyltransferase activity are: pH optimum 8.0, Km values for orotate and PRPP of 4.5 and 5.4µM, respectively. Orotidine-5'-phosphate decarboxylase activity is optimal between pH 7.2 and 8.5, the Km value for orotidine-5'-phosphate 2 µM.