Comparison of fluorescence-based semi-automated genotyping of multiple microsatellite loci with autoradiographic techniques.

The practical application of highly efficient fluorescence-based methods for the semi-automated genotyping of polymerase chain reaction-based microsatellite markers will depend on the development of robust protocols that provide accurate and reproducible data. In the present report we compare the accuracy of a fluorescence-based protocol with a benchmark radiolabeling method that depends on a known sequence ladder or amplified DNA from reference individuals for sizing by autoradiography. Three microsatellite markers, IGF1 (mfd 1), D4S174 (mfd 59), and D5S211 (mfd 154), with products overlapping in size were each labeled with a different fluorophore and run simultaneously with an internal size standard in a single electrophoretic lane. The size of each allele was compared for these markers by using both techniques for five larger CEPH families (884, 1331, 1332, 1333, and 1362). Of 462 possible alleles, four discrepancies (0.8%) were identified when the two approaches were compared. We conclude that the fluorescence-based protocol is at least as accurate as the standard radiolabeling technique since none of the sizing errors arose as a result of the fluorescence-based technique. We describe the adaptation of this fluorescence-based protocol to the simultaneous analysis of up to 24 microsatellite loci per electrophoretic lane. These highly accurate and efficient semi-automated techniques will be useful in high-resolution genomic analyses.

Developmental regulation of surfactant-associated proteins in rabbit fetal lung in vivo.

The developmental regulation of the rabbit surfactant-associated proteins, SP-A, SP-B, and SP-C, was investigated using Northern blot analysis. These proteins comprise approximately 10% by weight of pulmonary surfactant, a lipoprotein secreted by type II cells that reduces surface tension at the air-alveolar interface. SP-A mRNA and SP-B mRNA were first detected in rabbit fetal lung at day 24 of gestation (term = 31 days), i.e., approximately two days prior to the appearance of lamellar bodies within differentiated alveolar type II cells. The relative abundance of SP-B mRNA detected on day 24 of gestation was greater than that of SP-A mRNA. Fetal lung SP-A mRNA and SP-B mRNA levels increased rapidly during the remainder of gestation, reaching a maximum at day 31 of gestation. The relative concentrations of SP-A mRNA and SP-B mRNA were decreased in day 2 neonatal and adult lung tissues when compared to the levels present in fetal lung tissue late in gestation. A 0.5-kb rabbit SP-C cDNA was generated using the reverse transcriptase-polymerase chain reaction and was found to have high sequence homology to the human and rat SP-C cDNA nucleotide sequences. The predicted amino acid sequence for the rabbit SP-C cDNA revealed strong conservation of a hydrophobic region close to the amino terminus of the SP-C protein. Fetal lung SP-C mRNA was detected at day 19 of gestation, the earliest time point examined in this study. SP-C mRNA levels gradually increased in fetal lung tissue until day 28 of gestation and then remained level throughout the remainder of gestation and in the day 2 neonatal and adult rabbit lung tissue. These results suggest that the developmental pattern of induction of mRNA for the surfactant-associated proteins, SP-A, SP-B, and SP-C, differ from each other and are different in several respects from the developmental patterns observed in fetal lung tissue of the rat and human species.

Marfan syndrome caused by a recurrent de novo missense mutation in the fibrillin gene.

Marfan syndrome is an inherited disorder of connective tissue manifested in the ocular, skeletal and cardiovascular systems. It is inherited as an autosomal dominant with high penetrance, but has great clinical variability. Linkage studies have mapped the Marfan locus to chromosome 15q15-21.3. There have been no reports of genetic heterogeneity in the syndrome. Following the identification of fibrillin (a glycoprotein component of the extracellular microfibril), immunohistopathological quantification of the protein in skin and fibroblast culture, and examination of fibrillin synthesis, extracellular transport, and incorporation into the extracellular matrix (D. M. Milewicz, R.E.P., E. S. Crawford and P. H. Byers, manuscript in preparation) have demonstrated abnormalities of fibrillin metabolism in most patients. A portion of the complementary DNA encoding fibrillin has been cloned and mapped by in situ hybridization to chromosome 15. Here we report that the fibrillin gene is linked to the Marfan phenotype (theta = 0.00; logarithm of the odds (lod) = 3.9) and describe a de novo missense mutation in the fibrillin gene in two patients with sporadic disease. We thus implicate fibrillin as the protein defective in patients with the Marfan syndrome.

Epitope mapping of monoclonal antibodies against caldesmon and their effects on the binding of caldesmon to Ca++/calmodulin and to actin or actin-tropomyosin filaments.

The effects of monoclonal anti-caldesmon antibodies, C2, C9, C18, C21, and C23, on the binding of caldesmon to F-actin/F-actin-tropomyosin filaments and to Ca++/calmodulin were examined in an in vitro reconstitution system. In addition, the antibody epitopes were mapped by Western blot analysis of NTCB (2-nitro-5-thiocyanobenzoic acid) and CNBr (cyanogen bromide) fragments of caldesmon. Both C9 and C18 recognize an amino terminal fragment composed of amino acid residues 19 to 153. The C23 epitope lies within a fragment ranging from residues 230 to 386. Included in this region is a 13-residue repeat sequence. Interestingly this repetitive sequence shares sequence similarity with a sequence found in nuclear lamin A, a protein which is also recognized by C23 antibody. Therefore, it is likely that the C23 epitope corresponds to this 13-residue repeat sequence. A carboxyl-terminal 10K fragment contains the epitopes for antibodies C2 and C21. Among these antibodies, only C21 drastically inhibits the binding of caldesmon to F-actin/F-actin-tropomyosin filaments and to Ca++/calmodulin. When the molar ratio of monoclonal antibody C21 to caldesmon reached 1.0, a maximal inhibition (90%) on the binding of caldesmon to F-actin filaments was observed. However, it required double amounts of C21 antibody to exhibit a maximal inhibition of 70% on the binding of caldesmon to F-actin-tropomyosin filaments. These results suggest that the presence of tropomyosin in F-actin enhances caldesmon's binding. Furthermore, C21 antibody also effectively inhibits the caldesmon binding to Ca++/calmodulin. The kinetics of C21 inhibition on caldesmon's binding to Ca++/calmodulin is very similar to the inhibition obtained by preincubation of caldesmon with free Ca++/calmodulin. This result suggests that there is only one Ca++/calmodulin binding domain on caldesmon and this domain appears to be very close to the C21 epitope. Apparently, the Ca++/calmodulin-binding domain and the actin-binding domain are very close to each other and may interfere with each other. In an accompanying paper, we have further demonstrated that microinjection of C21 antibody into living chicken embryo fibroblasts inhibit intracellular granule movement, suggesting an in vivo interference with the functional domains [Hegmann et al., 1991: Cell Motil. Cytoskeleton 20:109-120].

Monoclonal antibodies against caldesmon, a Ca++/calmodulin- and actin-binding protein of smooth muscle and nonmuscle cells.

Monoclonal antibodies, C2, C9, C18, C21 and C23, against chicken gizzard caldesmon have been prepared and characterized. These antibodies reacted with gizzard caldesmon (150 KDa) by enzyme-linked immunosorbent assay and protein immunoblotting. Immunofluorescence microscopy with these antibodies on cultured gizzard cells showed strong stress fiber and membrane ruffle stainings. Surprisingly, in addition to these cytoplasmic staining patterns, the C23 antibody also stained nuclei in these cells. Preabsorption of C23 with purified caldesmon abolished the staining of stress fibers and membrane ruffles as well as the staining of nuclei, suggesting that a common epitope existed in both gizzard caldesmon and the nuclear protein. Western blot analysis on the cell extract of chicken embryo fibroblast (CEF) showed that antibodies C2, C9, C18 and C21 recognized a nonmuscle caldesmon (66 KDa), whereas C23 reacted with a protein (60 KDa) different from nonmuscle caldesmon. Antibody C21 also crossreacted with a nonmuscle caldesmon (80 KDa) in normal rat kidney (NRK) cells, with a nonmuscle caldesmon (78 KDa) in human cells, and with a nonmuscle caldesmon (72 KDa) in gerbil fibroma cells. This antibody had broad-species specificity. Immunofluorescent staining of CEF cells with antibodies C2, C9, C18 and C21 showed some stress fibers and ruffles, but mostly diffuse staining. Antibody C23 crossreacted with 62 KDa and 55 KDa proteins in NRK cells, 63 KDa and 55 KDa proteins in gerbil fibroma cells and 66 KDa and 56 KDa proteins in human bladder carcinoma cells. These polypeptides were identified as nuclear lamins A and C by an anti-lamin antibody in immunoblots and two-dimensional gel analysis. Like the nuclear lamins, the C23 antigens also underwent a reversible disassembly during mitosis, as detected by double-label immunofluorescence with C23 antibody and a polyclonal anti-tubulin antibody. Tropomyosin-enriched microfilaments isolated from fibroblastic and epithelial types of NRK cells by monoclonal anti-tropomyosin antibody contained an 80 KDa protein, which had the heat-resistant property of caldesmon. The polyclonal antiserum generated against this 80 KDa protein showed a crossreactivity with purified gizzard caldesmon and vice versa. The amount of this nonmuscle caldesmon associated with the microfilaments of Kirstein virus-transformed NRK cells was greatly decreased.