Abnormal RNA expression of 11p15 imprinted genes and kidney developmental genes in Wilms' tumor.

Wilms' tumor (WT) is caused by abnormal development of embryonal kidney cells. WT cells are frequently affected by deletions or functional inactivation of maternal alleles at chromosome 11p15, which indicates that the loss of maternally expressed genes in this region plays an important role in WT pathogenesis. Maternally expressed genes indeed exist within an imprinted region at 11p15.5. Among these, BWR1C is highly expressed in fetal but not in adult kidney, which suggests that it may fulfil an important role in kidney development. Here, we demonstrate that the lack of BWR1C expression is common in WT. Its homology with the proapoptotic gene TDAG51 suggests that the loss of BWR1C expression may be relevant in WT development. In addition, the analysis of the expression of other 11p15 imprinted genes and kidney-developmentally regulated genes indicates that IGF2 overexpression, inappropriate coexpression of RET and GDNF and, in some cases, down-regulation of CDKN1C may also play an important role in the pathogenesis of WT. Our results add new elements to the understanding of the biological basis of WT, which may have implications for WT diagnosis and therapy.

Gain of imprinting at chromosome 11p15: A pathogenetic mechanism identified in human hepatocarcinomas.

Genomic imprinting is a reversible condition that causes parental-specific silencing of maternally or paternally inherited genes. Analysis of DNA and RNA from 52 human hepatocarcinoma samples revealed abnormal imprinting of genes located at chromosome 11p15 in 51% of 37 informative samples. The most frequently detected abnormality was gain of imprinting, which led to loss of expression of genes present on the maternal chromosome. As compared with matched normal liver tissue, hepatocellular carcinomas showed extinction or significant reduction of expression of one of the alleles of the CDKN1C, SLC22A1L, and IGF2 genes. Loss of maternal-specific methylation at the KvDMR1 locus in hepatocarcinoma correlated with abnormal expression of CDKN1C and IGF2, suggesting a function for KvDMR1 as a long-range imprinting center active in adult tissues. These results point to the role of epigenetic mechanisms leading to loss of expression of imprinted genes at chromosome region 11p15 in human tumors.

Differential actin organization by vinculin isoforms: implications for cell type-specific microfilament anchorage.

Vinculin is found in all adherens junctions, while metavinculin, a larger splice variant, is coexpressed with vinculin only in smooth and cardiac muscle. To understand the significance of metavinculin expression, we compared ligand binding between turkey vinculin and metavinculin. Residues 1-258 were found essential for head-tail interactions in both proteins. The tail domains (VT and MVT, respectively) both bind to F-actin. However, while VT bundles F-actin, MVT generates highly viscous F-actin webs. In transfected PtK2 cells, VT causes F-actin needles or coils, while MVT-expressing cells display a diffuse F-actin distribution. Thus, the MVT-specific insert induces an F-actin supraorganization different from the VT-based form, suggesting that metavinculin has a specific role in muscle.

Transcriptional map of 170-kb region at chromosome 11p15.5: identification and mutational analysis of the BWR1A gene reveals the presence of mutations in tumor samples.

Chromosome region 11p15.5 harbors unidentified genes involved in neoplasms and in the genetic disease Beckwith-Wiedemann syndrome. The genetic analysis of a 170-kb region at 11p15.5 between loci D11S601 and D11S679 resulted in the identification of six transcriptional units. Three genes, hNAP2, CDKN1C, and KVLQT1, are well characterized, whereas three genes are novel. The three additional genes were designated BWR1A, BWR1B, and BWR1C. Full-length cDNAs for these three genes were cloned and nucleotide sequences were determined. While our work was in progress, BWR1C cDNA was described as IPL [Qian, N., Franck, D., O'Keefe, D., Dao, D. , Zhao, L., Yuan, L., Wang, Q., Keating, M., Walsh, C. & Tycko, B. (1997) Hum. Mol. Genet. 6, 2021-2029]. The cloning and mapping of these genes together with the fine mapping of the three known genes indicates that the transcriptional map of this region is likely to be complete. Because this region frequently is altered in neoplasms and in the genetic disease Beckwith-Wiedemann syndrome, we carried out a mutational analysis in tumor cell lines and Beckwith-Wiedemann syndrome samples that resulted in the identification of genetic alterations in the BWR1A gene: an insertion that introduced a stop codon in the breast cancer cell line BT549 and a point mutation in the rhabdomyosarcoma cell line TE125-T. These results indicate that BWR1A may play a role in tumorigenesis.

Refined subchromosomal location of 21 expressed sequence tags from unknown genes at region 11p15.

Twenty-one expressed sequence tags (ESTs) belonging to unidentified transcripts were mapped to 9 intervals at 11p 15.5-p15.3. Thirteen were mapped to a region of an estimated size of 7 Mb with the help of a YAC contig. The remaining eight were mapped outside this region, and the centromeric or telomeric position of the ESTs relative to the YAC contig was defined with the help of a cellular hybrid containing a derivative chromosome 11 with a break-point within the region covered by the contig. The average size of the intervals, where the ESTs were mapped, was estimated to be 0.3-0.4 Mb. The refinement of the chromosomal location of these ESTs could be of great help in the identification of potential candidate genes for disease locus mapping at 11p15.

A model relating protein osmotic pressure to the stiffness of the cross-bridge components and the contractile force of skeletal muscle.

We have modeled the effect of protein osmotic pressure on the orientation of the monomer in F-actin, in tropomyosin-F-actin, in the myosin subfragment-1 decorated F-actin and in the myosin subfragment-1 decorated tropomyosin-F-actin. According to the model, at the physiological protein osmotic pressure (18 kPa), the elastic moduli by bending of the monomer in F-actin and in tropomyosin-F-actin are calculated to be 4.74 MPa and 5.8 MPa, respectively. The elastic moduli by bending of the monomer in the myosin subfragment-1 decorated F-actin and in the myosin subfragment-1 decorated tropomyosin-F-actin are calculated to be 22MPa and 22.3MPa, respectively. These latter values are in excellent agreement with the values of the elastic moduli by stretching found for the fibres of frog and rabbit muscle. We have also calculated that, at the physiological protein osmotic pressure, the myosin subfragment-1 decorated F-actin rigor complex can develop a force of 3.96 pN, a force correctly oriented to promote the sliding of the actin filament toward the center of the sarcomere. The magnitude of this force is comparable to that reported for intact skeletal muscle. In contrast, the myosin subfragment-1 decorated tropomyosin-F-actin rigor complex develops a much smaller driving force, that favours relaxation. Apparently tropomyosin uncouples the osmotic and the mechanical event. It is proposed that the elastic energy for muscle contraction is provided by protein osmotic pressure.

The stiffness of the crossbridge is a function of the intrinsic protein osmotic pressure generated by the crossbridge itself.

A model is presented that makes it possible to determine the stiffness of the crossbridge from protein osmotic stress experiments. The model was elaborated while studying the osmotic properties of F-actin and of myosin subfragment-1 F-actin. These studies showed that the elastic modulus by bending of the monomer is directly related to the intrinsic protein osmotic pressure of the system. At a protein osmotic pressure of 1.8 x 10(5) dynes/cm2, the physiological protein osmotic pressure of frog skeletal muscle, it was found that the elastic moduli by bending of the monomer in F-actin and in the myosin subfragment-1 decorated F-actin are 6.5 X 10(7) and 3.3 X 10(8) dynes/cm2, respectively. The value of the elastic modulus by bending of the monomer in the myosin subfragment-1 decorated F-actin compares favorably with the values of the elastic modulus by stretching determined in skeletal muscle fibres.

Intramolecular interactions regulate serine/threonine phosphorylation of vinculin.

Using protein kinase C, we have studied the influence of intramolecular interactions on phosphorylation in vinculin. We show that vinculin and its 90 kDa head and 29/27 kDa tail fragments, generated by V8 proteolytic cleavage, are differentially phosphorylated. While intact vinculin and the isolated head domain are only weakly labelled, the isolated tail fragment is much more strongly phosphorylated. In the presence of the tail, the head is fully protected from the kinase. These data are consistent with our observation that native vinculin is primarily phosphorylated within the tail domain and suggest a function of vinculin phosphorylation in the regulation of the vinculin conformation.

Osmotic properties of myosin subfragment 1: implications of the mechanism of muscle contraction.

The osmotic behavior of myosin subfragment 1 was studied at 22 degrees C and pH 7.45 in 0.1 m KCl, 2 mm MgCl2, and 10 mm triethanolamine or in 25 mm phosphate, 2 mm MgCl2, and 2 mm MgADP. It was found that, in 0.1 m KCl, myosin subfragment 1 behaved as a spheroidal particle, with an average diameter of 8.09 nm, composed of two myosin subfragment 1 molecules. The lower limit of the thermodynamic dimerization constant was estimated to be 3.5 x 10(4) M-1. Above 5 mm as monomer, myosin subfragment 1 departed from the behavior expected of a dimeric spheroidal model because of the onset of a "hydration force." This force measured at the contact distance between particles equals 2.18 x 10(7) dynes/cm2 and falls off exponentially with a decay distance of 0.27 nm. In 25 mm orthophosphate, myosin subfragment 1, with an increase in the protein osmotic pressure, shifted from the behavior of a sphere to that of a cylinder. Between 1 x 10(5) and 4 x 10(5) dynes/cm2, the behavior of myosin subfragment 1 was different in the presence and in the absence of MgADP. In particular, at 1.8 x 10(5) dynes/cm2, the protein osmotic pressure in frog muscle, myosin subfragment 1 behaved as a sphere of 3.21-nm radius in the presence of MgADP and as a cylinder with a length to diameter ratio of 2.07 in the absence of MgADP. Under the solution conditions used in this work, S1 never behaved as a fully extended particle.

Osmotic properties of the calcium-regulated actin filament.

The diameter of the actin filament decreases with an increase of the protein osmotic pressure. This phenomenon is accompanied by a decrease of the angle (alpha) formed between the long axis of the actin monomer and the pointed end of the filament axis. At 1.8 x 10(5) dyn/cm2 (the protein osmotic pressure in frog muscle) the diameter is 8.34 nm and the angle (alpha) is 61.5 degrees. The interfilament distance of tropomyosin-decorated actin filaments, at a set of different osmotic pressures, is larger than that of F-actin filaments. This suggests that the two tropomyosin helices project out of the contour of the actin filament. The tropomyosin-decorated actin filament is more rigid than F-actin. At 1.8 x 10(5) dyn/cm2, the angle (alpha) is 76.4 degrees, as compared to the value of 61.5 degrees for F-actin. The interfilament distance of troponin-tropomyosin-decorated actin filaments is sensitive to Ca2+: in the physiological range of protein osmotic pressure it decreases from 13.3 nm, in the presence of 2 mM EGTA, to 12.2 nm in the presence of 0.2 mM CaCl2. Two alternative models are proposed to explain the decrease in interfilament distance. (a) Calcium shifts tropomyosin along the actin monomer, toward the filament axis (the classical model). (b) Calcium releases the rigidity of the tropomyosin-decorated filament and restores the original plasticity of F-actin. The consequent decrease of the angle (alpha) brings the tropomyosin helices nearer to the filament axis, without any real movement of tropomyosin along the actin monomer.

Actin may contribute to the power stroke in the binary actomyosin system.

At the physiological protein osmotic pressure, the angle formed between the long axis of the actin monomer and the pointed end of the filament axis is roughly 61 degrees in F-actin and about 90 degrees in the myosin subfragment 1--decorated F-actin. This implies that, in the course of the contractile cycle, actin itself contributes, by about 4 nm, to the displacement of the actin filament toward the center of the sarcomer.

alpha-Actinin from chicken gizzard: at low temperature, the onset of actin-gelling activity correlates with actin bundling.

The effect of alpha-actinin from chicken gizzard on the properties of F-actin solutions at 37 degrees C and at 4 degrees C was investigated. Beside the well-known increase of the gelling activity of alpha-actinin, it was found that lowering temperature to 4 degrees C: (a) modifies the shape of the alpha-actinin-F-actin binding isotherm; (b) increases the light scattering of the alpha-actinin-F-actin mixtures; (c) induces the formation of ribbons and bundles of F-actin. It was also observed that, by warming to 37 degrees C, the bundles of F-actin formed at 4 degrees C were dissociated into quasi-parallel actin filaments running at a distance of 25-42 nm from each other. On subsequent cooling to 4 degrees C, these parallel filaments were rapidly assembled into bundles. As at 37 degrees C, alpha-actinin displays a potent gelling activity on bundles but not on filaments of actin, and as the gelling activity at 4 degrees C is accompanied by the formation of actin bundles, it is concluded that actin bundling is a necessary condition to promote the actin-gelling activity.

Osmotic stress is the main determinant of the diameter of the actin filament.

The diameter of the actin filament is influenced by osmotic stress, being 9.0 nm at 1 x 10(5) dynes/cm2 and 6.8 nm at 9.00 x 10(6) dynes/cm2. At 1.81 x 10(5) dynes/cm2, the protein osmotic pressure in frog muscle, the diameter is 7.95 nm. The diameter of the tropomyosin-decorated actin filament is also influenced by osmotic stress even though, at the low pressures (up to 2 x 10(5) dynes/cm2), the decorated filament is significantly more resistant to compression than the undecorated actin filament.

Diffusion hindrance and geometry of filament crossings account for the complex interactions of F-actin with alpha-actinin from chicken gizzard.

The interaction of alpha-actinin from chicken gizzard with F-actin is quite complex. The apparent dissociation constant, C, increases with the increase of actin concentration according to the following expression: C = Ko + a[actin] - c[actin]5/2. At pH 7.5 and 37 degrees C, in the presence of 0.1 M KCl and 2 mM MgCl2, the dissociation constant at infinite actin dilution, Ko, is 2.17 microM. The binding of alpha-actinin to actin is related by the term a[actin] to the diffusion of actin filaments and by the term c[actin]5/2 to the crossing number concentration of the F-actin network. Especially at low actin concentration, the binding of alpha-actinin to actin is increased by gelsolin, which fragments actin filaments and increases their diffusion. The different binding isotherms of alpha-actinin to actin filaments and to actin bundles are discussed.

In-frame deletion of von Willebrand factor A domains in a dominant type of von Willebrand disease.

von Willebrand disease (vWD), the most common inherited bleeding disorder in humans, is very heterogeneous and has been classified into several subtypes. Missense mutations have been found to be responsible for the dominant type II vWD, characterized by qualitative abnormalities affecting von Willebrand factor (vWF) function. The breakpoints of a heterozygous vWF gene deletion (31 Kb), occurring 'de novo' in a patient with a variant of type II vWD, were localized to introns 25 and 34 and sequenced. An Alu repeat in intron 25 was interrupted between the transcriptional boxes A and B. The new junction present in the abnormal von Willebrand factor mRNA was sequenced after reverse transcription of platelet RNA. The codon 1104 (Cys) is followed in frame by the mutated codon 1926 (Cys to Arg), thus removing the complete A domains, found in a wide variety of genes and characterized by independent assembly 'in vitro'. We propose that the abnormal vWF, which carries intact protein domains responsible for vWF dimer and multimer formation, makes ineffective interactions with the normal molecules in the biosynthetic process, causing the dominant type II phenotype through a novel mechanism.

Preferential binding of alpha-actinin to actin bundles.

At 37 degrees C, the alpha-actin-F-actin binding isotherm is anomalous. In 6.7% polyethylene glycol 6000, concomitantly with the formation of actin bundles, the binding isotherm becomes hyperbolic (Kdiss. = 11.3 microM). alpha-Actinin increases the rigidity of the networks formed by actin bundles in polyethylene glycol and by paracrystalline actin in 16 mM MgCl2 but not by F-actin. It is proposed that in the cell alpha-actinin functions are mostly carried on by interaction with actin bundles.

Characterization of the pseudogenic and genic homologous regions of von Willebrand factor.

The homologous pseudogenic and genic regions of von Willebrand factor (vWF) were studied in DNA from a patient with homozygous deletion of vWF genes and compared with a normal control. This analysis indicates informative restriction patterns for the investigation of restriction fragment length polymorphisms (RFLPs) and gene lesions, and for molecular cloning. A useful new genic XbaI RFLP was found and characterized. A large BgIII fragment of the pseudogenic region was cloned and mapped, and single sequences (9 kb) were used as probes. Corresponding genic and pseudogenic fragments, which contain exons 23-28, and specific restriction patterns were identified, including a new polymorphic TaqI site that was mapped in the gene. A cloned fragment contains the 5' boundary of the pseudogene and recognizes an additional and unknown homologous sequence in the genome. The chromosomal localization of the vWF pseudogene and of the breakpoint cluster region (BCR) gene were compared by 'in situ' hybridization: overlapping patterns were detected. The cloning, characterization and mapping of the pseudogenic region improves the analysis of this portion of chromosome 22 affected by several somatic and constitutional alterations, and also of the corresponding genic region on chromosome 12.

A recurrent missense mutation (Arg----Gln) and a partial deletion in factor VIII gene causing severe haemophilia A.

The presence of gene lesions in coagulation factor VIII (FVIII) gene was investigated in 70 Italian patients severely affected by haemophilia A. cDNA probes specific for exons 14-26 of the FVIII gene were used. In two related patients a gene deletion removes exon 26, a gene lesion similar to that described previously in a British haemophiliac. In exon 24 a C to T transition in the reverse complement strand causes a missense mutation in the coding strand (CGA----CAA, 2209 Arg----Gln). The mutation is located in a very conserved FVIII homology region and severely reduces FVIII activity. By restriction analysis and hybridizations with oligonucleotide probes this gene alteration was found in two unrelated haemophiliacs and in their relatives.