We have determined the complete primary structure (8031 base pairs) of an infectious clone of cauliflower mosaic virus strain CM1841. The sequence was obtained using the strategy of cloning shotgun restriction fragments in the sequencing vector M13mp7. Comparison of the CM1841 sequence with that published for another caMV strain (Strasbourg) reveals 4.4% changes, mostly nucleotide substitutions with a few small insertions and deletions. The six open reading frames in the sequence of the Strasbourg isolate are also present in CM1841.

In the liver of oviparous vertebrates vitellogenin gene expression is controlled by estrogen. The nucleotide sequence of the 5' flanking region of the Xenopus laevis vitellogenin genes A1, A2, B1 and B2 has been determined. These sequences have been compared to each other and to the equivalent region of the chicken vitellogenin II and apo-VLDLII genes which are also expressed in the liver in response to estrogen. The homology between the 5' flanking region of the Xenopus genes B1 and B2 is higher than between the corresponding regions of the other closely related genes A1 and A2. Four short blocks of sequence homology which are present at equivalent positions in the vitellogenin genes of both Xenopus laevis and chicken are characterized. A short sequence with two-fold rotational symmetry (GGTCANNNTGACC) was found at similar positions upstream of the five vitellogenin genes and is also present in two copies close to the 5' end of the chicken apo-VLDLII gene. The possible functional significance of this sequence, common to liver estrogen-responsive genes, is discussed.

We cloned and characterized a novel member of the tenascin family of extracellular matrix proteins - the murine orthologue of zebrafish tenascin-W. Full-length recombinant tenascin-W was expressed and purified from mammalian cell cultures. Rotary shadowing followed by electron microscopy showed that tenascin-W forms hexabrachions. We studied its expression during development and in the adult by immunohistochemistry, in situ hybridization and immunoblotting. Tenascin-W is expressed during palate formation, osteogenesis and smooth muscle development. In the adult, tenascin-W is found in the kidney, cardiac semilunar valves, corneal limbus and periosteum. Tenascin-W and tenascin-C expression overlap in many of these areas. Bone-morphogenic-protein-2 treated C2C12 cells secrete tenascin-W and are able to adhere to and to extend actin-rich processes on a tenascin-W substratum. In vitro, cells bind to tenascin-W in an RGD-dependent manner. This adhesion is increased by transfection of α8 integrin, which localizes with tenascin-W in the periosteum and kidney.

Tenascins represent a family of extracellular matrix glycoproteins with distinctive expression patterns. Here we have analyzed the most recently described member, tenascin-W, in breast cancer. Mammary tumors isolated from transgenic mice expressing hormone-induced oncogenes reveal tenascin-W in the stroma around lesions with a high likelihood of metastasis. The presence of tenascin-W was correlated with the expression of its putative receptor, a8 integrin. HC11 cells derived from normal mammary epithelium do not express a8 integrin and fail to cross tenascin-W-coated filters. However, 4T1 mammary carcinoma cells do express a8 integrin and their migration is stimulated by tenascin-W. The expression of tenascin-W is induced by BMP-2 but not by TGF-b1, though the latter is a potent inducer of tenascin-C. The expression of tenascin-W is dependent on p38 MAPK and JNK signaling pathways. Since preinflammatory cytokines also act through p38 MAPK and JNK signaling pathways, the possible role of TNF-a in tenascin-W expression was also examined. TNF-a induced the expression of both tenascin-W and tenascin-C, and this induction was p38 MAPK -and cyclooxygenase-dependent. Our results show that tenascin-W may be a useful diagnostic marker for breast malignancies, and that the induction of tenascin-W in the tumor stroma may contribute to the invasive behavior of tumor cells.

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Recombinant rat glia-derived nexin was expressed in insect cells using the baculovirus system. The kinetics for the inhibition of thrombin by this recombinant material were indistinguishable from those observed with natural glia-derived nexin and recombinant nexin expressed in yeast. In addition, the dependence of the rate of inactivation on the concentration of heparin was similar for the three preparations. At the optimal heparin concentration, the association rate constant was 330-fold higher than that observed in the absence of heparin. A putative heparin-binding site is found in glia-derived nexin between residues 71 and 86; heparin-binding sites are found in homologous regions of antithrombin III and heparin cofactor II. Lysines in this region were mutated to glutamates, and the kinetics for the inhibition of thrombin by mutant proteins were determined. Concurrent mutation of all seven lysines in this region (residues 71, 74, 75, 78, 83, 84, and 86) did not affect the rate constant for the association of glia-derived nexin with thrombin in the absence of heparin, but it resulted in complete loss of the heparin acceleration of the rate of association. Mutations of residues 83, 84, and 86 together also caused a marked decrease in the acceleration by heparin of the reaction between glia-derived nexin and thrombin. These results support the hypothesis that the heparin-binding sites of glia-derived nexin, antithrombin III, and heparin cofactor II are found in homologous regions of the molecules. Heparin was also found to potentiate the ability of wild-type glia-derived nexin to inhibit the thrombin-induced retraction of neurites from neuroblastoma NB2a cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Five initiation factors, eIF-2, eIF-3, eIF-4A, eIF-4B, and eIF-5, were purified from human HeLa cells. Methods of protein fractionation and assays for initiation factors which had been developed for rabbit reticulocytes were found to be suitable for HeLa factors. The initiation factors from HeLa cells are similar to or indistinguishable from the corresponding rabbit reticulocyte factors with respect to specific activities, molecular weights as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subunit structure. The molecular weight of eIF-3 particles from both species is about 410000 as determined by equilibrium sedimentation analytical centrifugation. The partial protease fragmentation patterns of corresponding proteins also are similar and indicate that the primary sequences of the factors are related in the two species. Antisera raised in goats against rabbit eIF-3 and human eIF-2, eIF-4A, and eIF-4B cross-react with the cognate factors from both species. On the basis of immunoblotting techniques, eIF-4A is highly conserved, eIF-2 alpha, eIF-3, and eIF-4B are somewhat less conserved, and eIF-2 beta is the least conserved of the proteins examined. The functional, structural, and immunological results are all consistent with the view that initiation factors from different mammalian cells are very similar.