Ovomucoid intervening sequences specify functional domains and generate protein polymorphism

Two thirds of the natural chicken ovomucoid gene has been sequenced, including all exons and the intron sequences surrounding all fourteen intron/ exon junctions. The junction sequences surrounding four of the introns are redundant; however, the sequences surrounding the other three introns contain no redundancies and thus the splicing sites at either end of these three introns are unambiguous. The splicing in all cases conforms to the GT-AG rule. The ovomucoid gene sequence around intron F can be used to predict the cause of an internal deletion polymorphism in the ovomucoid protein, which is an apparent error in the processing of the ovomucoid pre-mRNA. We also compare the structural organization of the ovomucoid gene with the ovomucoid protein sequence to examine theories of the evolution of ovomucoids as well as the origin of intervening sequences. This analysis suggests that the present ovomucoid gene evolved from a primordial ovomucoid gene by two separate intragenic duplications. Furthermore, sequence analyses suggest that introns were present in the primordial ovomucoid gene before birds and mammals diverged, about 300 million years ago. Finally, the positions of the introns within the ovomucoid gene support the theory that introns separate gene segments that code for functional domains of proteins and provide insight on the manner by which eucaryotic genes were constructed during the process of evolution.     

Quiescent human diploid fibroblasts. Common mechanism for inhibition of DNA replication in density-inhibited and serum-deprived cells

The mechanism for cessation of proliferation in density-inhibited quiescent human diploid fibroblasts (HDF) and serum-deprived quiescent HDF was compared in two ways. Density-inhibited HDF were fused to either replicating HDF or SV40-transformed HDF and DNA synthesis was measured in the resulting heterokaryons. DNA synthesis was inhibited in the replicating HDF nuclei in heterokaryons in a way that suggested that entry into S phase was blocked, but ongoing DNA synthesis was not inhibited. In contrast, DNA synthesis was induced in the quiescent nuclei in heterokaryons formed with SV40-transformed HDF. Previous experiments had shown that serum-deprived HDF also behave in this way in heterokaryons. To test this similarity further, we examined the inhibitory activity of cell membranes prepared from both types of quiescent HDF. We found that both types of quiescent HDF contain DNA synthesis-inhibitory activity that is (1) effective on replicating HDF; (2) ineffective on SV40-transformed HDF; (3) sensitive to heat and trypsin. Thus, these results support the hypothesis that both density-inhibited HDF and serum-deprived HDF share a common mechanism for arrest in G1 phase. They also suggest that a membrane-bound protein plays a role in the inhibition of DNA synthesis in quiescent HDF.     

TGF-β1 modifications in nuclear matrix proteins of osteoblasts during differentiation

Nuclear matrix protein (NMP) composition of osteoblasts shows distinct two-dimensional gel electrophoretic profiles of labeled proteins as a function of stages of cellular differentiation. Because NMPs are involved in the control of gene expression, we examined modifications in the representation of NMPs induced by TGF-β1 treatment of osteoblasts to gain insight into the effects of TGF-β on development of the osteoblast phenotype. Exposure of proliferating fetal rat calvarial derived primary cells in culture to TGF-β1 for 48 h (day 4–6) modifies osteoblast cell morphology and proliferation and blocks subsequent formation of mineralized nodules. Nuclear matrix protein profiles were very similar between control and TGF-β–treated cultures until day 14, but subsequently differences in nuclear matrix proteins were apparent in TGF-β–treated cultures. These findings support the concept that TGF-β1 modifies the final stage of osteoblast mineralization and alters the composition of the osteoblast nuclear matrix as reflected by selective and TGF-β–dependent modifications in the levels of specific nuclear matrix proteins. The specific changes induced by TGF-β in nuclear matrix associated proteins may reflect specialized mechanisms by which TGF-β signalling mediates the alterations in cell organization and nodule formation and/or the consequential block in extracellular mineralization. J. Cell. Biochem. 69:291–303, 1998. © 1998 Wiley-Liss, Inc.     

Paradoxical effects of a stress signal on pro- and anti-apoptotic machinery in HTLV-1 tax expressing cells

Adult T-cell leukemia (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) are associated with Human T-cell lymphotropic virus type 1 (HTLV-1) infection. The viral transactivator, Tax is able to mediate the cell cycle progression by targeting key regulators of the cell cycle such as p21/waf1, p16/ink4a, p53, cyclins D1-3/cdk complexes, and the mitotic spindle checkpoint MAD apparatus, thereby deregulating cellular DNA damage and checkpoint control. Genome expression profiling of infected cells exemplified by the development of DNA microarrays represents a major advance in genome-wide functional analysis. Utilizing cDNA microarray analysis, we have observed an apparent opposing and paradoxical regulatory network of host cell gene expression upon the introduction of DNA damage stress signal. We find the apparent induction of cell cycle inhibitors, and pro- as well as anti-apoptotic gene expression is directly linked to whether cells are at either G1, S, or G2/M phases of the cell cycle. Specifically, a G1/S block is induced by p21/waf1 and p16/ink4a, while pro-apoptotic expression at S, and G2/M is associated with caspase activation, and anti-apoptotic gene expression is associated with up regulation of Bcl-2 family member, namely bfl-1 gene. Therefore, the microarray results indicating expression of both pro- and anti-apoptotic genes could easily be explained by the particular stage of the cell cycle. Mechanism and the functional outcome of induction for both pathways are discussed.     

The Wnt antagonist secreted frizzled-related protein-1 is a negative regulator of trabecular bone formation in adult mice

Previous studies have associated activation of canonical Wnt signaling in osteoblasts with elevated bone formation. Here we report that deletion of the murine Wnt antagonist, secreted frizzled-related protein (sFRP)-1, prolongs and enhances trabecular bone accrual in adult animals. sFRP-1 mRNA was expressed in bones and other tissues of +/+ mice but was not observed in -/- animals. Despite its broad tissue distribution, ablation of sFRP-1 did not affect blood and urine chemistries, most nonskeletal organs, or cortical bone. However, sFRP-1-/- mice exhibited increased trabecular bone mineral density, volume, and mineral apposition rate when compared with +/+ controls. The heightened trabecular bone mass of sFRP-1-/- mice was observed in adult animals between the ages of 13-52 wk, occurred in multiple skeletal sites, and was seen in both sexes. Mechanistically, loss of sFRP-1 reduced osteoblast and osteocyte apoptosis in vivo. In addition, deletion of sFRP-1 inhibited osteoblast lineage cell apoptosis while enhancing the proliferation and differentiation of these cells in vitro. Ablation of sFRP-1 also increased osteoclastogenesis in vitro, although changes in bone resorption were not observed in intact animals in vivo. Our findings demonstrate that deletion of sFRP-1 preferentially activates Wnt signaling in osteoblasts, leading to enhanced trabecular bone formation in adults.     

The Effects of Silicone Fluid Additives and Silicone Elastomer Matrices on Barnacle Adhesion Strength

Barnacle adhesion strength was used to screen seventy-seven polydimethylsiloxane elastomeric coatings for fouling-release properties. The test coatings were designed to investigate the effect on barnacle adhesion strength of silicone fluid additive type, additive location, additive molecular weight, additive loading level, mixtures of additives, coating matrix type and coating fillers. The type of silicone fluid additive was the primary controlling factor in barnacle fouling-release. The type of silicone matrix in which the fluid resided was found to alter the effect on fouling-release. Two PDMS fluids, DMSC15 and DBE224, significantly reduced the adhesion strength of barnacles compared to unmodified elastomers. Optimum fouling-release performance was dependent on the interaction of fluid type and elastomeric matrix.     

A genetic map of tetraploid <Emphasis Type="Italic">Paspalum notatum</Emphasis> Flügge (bahiagrass) based on single-dose molecular markers

Paspalum notatum Flügge is a warm-season forage grass with mainly diploid (2n = 20) and autotetraploid (2n = 40) representatives. Diploid races reproduce sexually and require crosspollination due to a self-incompatible mating system, while autotetraploids reproduce by aposporous apomixis. The objectives of this work were to develop a genetic linkage map of Paspalum notatum Flügge at the tetraploid level, identify the linkage/s group/s associated with apomixis and carry out a general characterization of its mode of inheritance. A pseudo test-cross F₁ family of 113 individuals segregating for the mode of reproduction was obtained by crossing a synthetic completely sexual tetraploid plant (Q4188) as female parent with a natural aposporous individual (Q4117) as pollen donor. Map construction was based on single-dose markers (SDAFs) segregating from both parents. Two linkage maps (female and male) were constructed. Within each map, homologous groups were assembled by detecting repulsion-phase linked SDAFs. Putative Q4188 and Q4117 homolog groups were identified by mapping shared single dose markers (BSDF). The Q4188 map consisted of 263 markers distributed on 26 co-segregation groups over a total genetic distance of 1.590.6 cM, while the Q4117 map contained 216 loci dispersed on 39 co-segregation groups along 2.265.7 cM, giving an estimated genome coverage of 88% and 83%, respectively. Seven and 12 putative homologous chromosomes were detected within Q4188 and Q4117 maps, respectively. Afterward, ten female and male homologous chromosomes were identified by mapping BSDFs. In the Q4117 map, a single linkage group was associated with apospory. It was characterized by restriction in recombination and preferential chromosome pairing. A BPSD marker mapping within this group allowed the detection of the female homolog and the putative four male groups of the set carrying apospory.