Overexpression and assembly of the herpes simplex virus type 1 helicase-primase in insect cells

Herpes simplex virus type 1 (HSV-1) encodes a helicase-primase that consists of three polypeptides encoded by the UL5, UL8, and UL52 genes (Crute, J.J., Tsurumi, T., Zhu, L., Weller, S.K., Olivo, P.D., Challberg, M.D., Mocarski, E.S., and Lehman, I.R. (1989) Proc. Natl. Acad, Sci, U.S.A. 86, 2186-2189). To obtain sufficient quantities of the enzyme for study, we have overexpressed the three genes using the baculovirus expression system. We find that the fully active enzyme can be assembled in vivo by triply infecting Spodoptera frugiperda SF9 cells with a baculovirus recombinant for each gene. The recombinant enzyme which we have purified to near homogeneity from the insect cells has a molecular weight of 270,000 and is composed of the three polypeptides encoded by the UL5, UL8, and UL52 genes. The enzyme possesses DNA-dependent ATPase, DNA-dependent GTPase, DNA helicase, and DNA primase activities that are essentially identical to the enzyme isolated from HSV-1-infected cells.     

Role of the Subchondral Vascular System in Endochondral Ossification: Endothelial Cells Specifically Derepress Late Differentiation in Resting Chondrocytesin Vitro

Endochondral ossification in growth plates proceeds through several consecutive steps of late cartilage differentiation leading to chondrocyte hypertrophy, vascular invasion, and, eventually, to replacement of the tissue by bone. It is well established that the subchondral vascular system is pivotal in the regulation of this process. Cells of subchondral blood vessels act as a source of vascular invasion and, in addition, release factors influencing growth and differentiation of chondrocytes in the avascular growth plate. To elucidate the paracrine contribution of endothelial cells we studied the hypertrophic development of resting chondrocytes from the caudal third of chick embryo sterna in co-culture with endothelial cells. The design of the experiments prevented cell-to-cell contact but allowed paracrine communication between endothelial cells and chondrocytes. Under these conditions, chondrocytes rapidly became hypertrophiedin vitroand expressed the stage-specific markers collagen X and alkaline phosphatase. This development also required signaling by thyroid hormone in synergy. Conditioned media could replace the endothelial cells, indicating that diffusible factors mediated this process. By contrast, smooth muscle cells, fibroblasts, or hypertrophic chondrocytes did not secrete this activity, suggesting that the factors were specific for endothelial cells. We conclude that endochondral ossification is under the control of a mutual communication between chondrocytes and endothelial cells. A finely tuned balance between chondrocyte-derived signals repressing cartilage maturation and endothelial signals promoting late differentiation of chondrocytes is essential for normal endochondral ossification during development, growth, and repair of bone. A dysregulation of this balance in permanent joint cartilage also may be responsible for the initiation of pathological cartilage degeneration in joint diseases.     

Herpes simplex-1 helicase-primase. Identification of two nucleoside triphosphatase sites that promote DNA helicase action.

Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase-primase comprised of the products of the UL5, UL8, and UL52 genes (Crute, J. J., and Lehman, I. R. (1991) J. Biol. Chem. 266, 4484-4488). A steady state kinetic analysis of the enzyme isolated from HSV-1-infected CV-1 cells or insect cells expressing the enzyme after infection with recombinant baculoviruses has shown it to possess two sites capable of hydrolyzing nucleoside triphosphates in a DNA-dependent manner. One site (Site I) hydrolyzes both ATP and GTP; the second (Site II) hydrolyzes only ATP. These two sites are contained within a subassembly of the helicase-primase formed by coexpression of the UL5 and UL52 genes in insect cells. Sites I and II are activated by separate DNA effector sites, both of which support DNA helicase action. These findings are likely to be of importance in understanding how helicases in general catalyze the unwinding of duplex DNA and, in particular, how the helicase-primase functions at the HSV-1 replication fork.     

Induction of proliferation or hypertrophy of chondrocytes in serum-free culture: the role of insulin-like growth factor-I, insulin, or thyroxine

In bone forming cartilage in vivo, cells undergo terminal differentiation, whereas most of the cells in normal articular cartilage do not. Chondrocyte hypertrophy can be induced also in vitro by diffusible signals. We have identified growth factors or hormones acting individually on 17-d chick embryo sternal chondrocytes cultured in agarose gels under strictly serum-free conditions. Insulin-like growth factor I or insulin triggered the first steps of chondrocyte maturation, i.e., cell proliferation and increased matrix deposition while the chondrocytic phenotype was maintained. However, cells did not progress to the hypertrophic stage. Proliferation and stimulated collagen production was preceded by a lag period, indicating that synthesis of other components was required before cells became responsive to insulin-like growth factor I or insulin. Very small amounts of FBS exerted effects similar to those of insulin-like growth factor I or insulin. However, FBS could act directly and elicited hypertrophy when constituting greater than 1% of the culture media. Basic FGF has been claimed to be the most potent chondrocyte mitogen, but had negligible effects under serum-free conditions. The same is true for PDGF, a major serum-mitogen. Under the direction of thyroxine, cells did not proliferate but became typical hypertrophic chondrocytes, extensively synthesizing collagen X and alkaline phosphatase.     

Cysteine in the triple-helical domain of one allelic product of the alpha 1(I) gene of type I collagen produces a lethal form of osteogenesis imperfecta

We studied tissue and cultured skin fibroblasts from a newborn with the lethal perinatal form of osteogenesis imperfecta born to a mother with the Marfan syndrome and her unrelated husband. Dermis from the infant was thinner and fibril diameter smaller than control; dermal fibroblastic cells had dilated endoplasmic reticulum. His fibroblasts in culture synthesized two different species of pro alpha 1(I) chains in about equal quantity. One chain was normal, the other contained cysteine within the triple-helical portion of the COOH-terminal cyanogen bromide peptide alpha 1(I)CB6. Molecules which contained two copies of the mutant chain formed alpha 1(I)-dimers linked through interchain disulfide bonds. Molecules which contained either one or two mutant chains were delayed in secretion and underwent excessive lysyl hydroxylation and hydroxylysyl glycosylation of all chains in the molecule, probably as a result of delayed triple-helix formation. Molecules containing either one or two copies of the mutant chain melted at 38 degrees C instead of 41 degrees C. The most likely explanation for these findings is that a cysteine is substituted for a glycine in the triple-helical domain of the products of one of the alpha 1(I) alleles. Such a substitution would interfere with triple-helix formation and stability and thus explain 1) the decreased melting temperature, 2) the increased post-translational modification, 3) the altered rate of secretion and accumulation of intracellular material, 4) the increased intracellular degradation of newly synthesized collagen, and 5) the decreased collagen production. Since neither parental cell strain produced the same mutant chain, the findings are best explained by a new mutation in one of the alpha 1(I) genes. The role of the uncharacterized "Marfan" gene in modifying the phenotype in this patient is unclear.     

p-HMW-collagen, a minor collagen obtained from chick embryo cartilage without proteolytic treatment of the tissue

The fragments of minor collagens of cartilages, called HMW and LMW, were isolated after pepsin treatment of sternal cartilages of young chickens and were shown to be entirely triple-helical molecules as judged by their circular dichroic spectra. Studies on renaturation kinetics of HMW suggested that the interchain disulfide bonds in HMW reside at one of the ends of the so-called long arm. Polyclonal antibodies against HMW were raised and affinity purified. These antibodies did not cross-react with type II collagen nor with other minor collagens such as LMW and 1 alpha, 2 alpha, 3 alpha collagen in native or denatured structure. The antibodies were used to identify HMW-related molecules which were synthesized by embryonic chick cartilages in vitro. Some of these molecules were secreted into the organ culture medium and could be recovered from it by ammonium sulfate precipitation. Polyacrylamide gel electrophoresis of this precipitate gave one band of high molecular weight which could be reduced to two bands migrating slightly faster than the alpha 1(II) chain when identified by immunoblotting. These bands could also be identified among about six radiolabelled polypeptides present in the ammonium sulfate precipitate of medium proteins when analysed by polyacrylamide gel electrophoresis followed by fluorography. The same polypeptides could be recovered from the medium by immunoprecipitation with anti-HMW antibodies. Their presence in cartilage tissue was shown by immunoblotting of material extracted from cartilage tissue and separated on polyacrylamide gels. We suggest that the protein containing these polypeptide chains represents the parent molecule of the peptic fragment HMW as it is synthesized in vivo and have designated it p-HMW-collagen.     

The glycosaminoglycan chain of decorin plays an important role in collagen fibril formation at the early stages of fibrillogenesis

Decorin is a multifunctional small leucine-rich proteoglycan involved in the regulation of collagen fibrillogenesis. In patients with a variant of Ehlers-Danlos syndrome, about half of the secreted decorin lacks the single glycosaminoglycan side chain. Notably, these patients have a skin-fragility phenotype that resembles that of decorin null mice. In this study, we investigated the role of glycanated and unglycanated decorin on collagen fibrillogenesis. Glycosaminoglycan-free decorin, generated by mutating Ser4 of the mature protein core into Ala (DCN-S4A), showed reduced inhibition of fibrillogenesis compared with the decorin proteoglycan. Interestingly, using a 3D matrix generated by decorin-null fibroblasts, an increase in fibril diameter was found after the addition of decorin, and even greater effects were observed with DCN-S4A. To avoid potential side effects of artificial tags, adenoviruses containing decorin and DCN-S4A were used to transduce decorin-null fibroblasts prior to matrix formation. Both molecules were efficiently incorporated into the matrix, with no changes in collagen composition and network formation, or altered expression of the related proteoglycan biglycan. Both decorin and DCN-S4A mutants increased the collagen fibril diameter, with the latter showing the most prominent effects. These data show that at early stages of fibrillogenesis, the glycosaminoglycan chain of decorin has a reducing effect on collagen fibril diameter.     

Translationally controlled tumour protein: A protein necessary for potyvirus intracellular multiplication that supports plant infection by unrelated viruses

The multifunctional protein translationally controlled tumour protein (TCTP) was previously identified as necessary for infection by the potyvirus pepper yellow mosaic virus. Using turnip mosaic virus (TuMV) as a model to study potyvirus biology, we confirmed that TCTP has a positive effect on virus infection. Living cell confocal microscopy demonstrated that TCTP colocalises with 6K2-tagged replication vesicles and with a perinuclear globular structure typically observed during potyvirus infection. Also, TCTP silenced protoplasts showed reduced virus accumulation, quantified by qRT-PCR, which suggests an effect on virus replication, translation or other intracellular process. Finally, TCTP silencing in plants reduced the accumulation of two species belonging to Orthotospovirus and a Begomovirus genus, which are not closely related to potyviruses. The results suggest that TCTP is a general susceptibility factor to several unrelated viruses.     

User guide for mapping-by-sequencing in Arabidopsis

Mapping-by-sequencing combines genetic mapping with whole-genome sequencing in order to accelerate mutant identification. However, application of mapping-by-sequencing requires decisions on various practical settings on the experimental design that are not intuitively answered. Following an experimentally determined recombination landscape of Arabidopsis and next generation sequencing-specific biases, we simulated more than 400,000 mapping-by-sequencing experiments. This allowed us to evaluate a broad range of different types of experiments and to develop general rules for mapping-by-sequencing in Arabidopsis. Most importantly, this informs about the properties of different crossing scenarios, the number of recombinants and sequencing depth needed for successful mapping experiments.