P64, a Novel Major Virion DNA-Binding Protein Potentially Involved in Condensing the Spodoptera frugiperda Ascovirus 1a Genome

We recently identified 21 structural proteins in the virion of Spodoptera frugiperda ascovirus 1a (SfAV1a), a virus with a large, double-stranded DNA genome of 157 kbp, which attacks species of the lepidopteran family Noctuidae. The two most abundant virion proteins were the major capsid protein and a novel protein (P64) of 64 kDa that contained two distinct domains not known previously to occur together. The amino-terminal half of P64 (residues 1 to 263) contained four repeats (a recently recognized motif with an unknown function) of a virus-specific two-cysteine adaptor. Adjoined to this, the carboxy-terminal half of P64 (residues 279 to 455) contained 14 copies of a highly basic, tandemly repeated motif rich in arginine and serine, having an 11- to 13-amino-acid consensus sequence, SPSQRRSTS(V/K)(A/S)RR, yielding a predicted isoelectric point of 12.2 for this protein. In the present study, we demonstrate by Southwestern analysis that SfAV1a P64 was the only virion structural protein that bound DNA. Additional electrophoretic mobility shift assays showed that P64 bound SfAV1a as well as non-SfAV1a DNA. Furthermore, we show through immunogold labeling of ultrathin sections that P64 is a component of virogenic stroma and appears to be progressively incorporated into the SfAV1a DNA core during virion assembly. As no other virion structural protein bound DNA and no basic DNA-binding proteins of lower mass are encoded by the SfAV1a genome or were identified by proteomic analysis, our results suggest that P64''s function is to condense the large genome of this virus and assist in packaging this genome into its virion.Following nucleic acid synthesis, viral genomes are condensed and packaged into virions by a variety of mechanisms (17, 23, 24, 30). Essential to this process is neutralization of the negative electrostatic charge on phosphate groups of the nucleotide backbone by positively charged ions that would otherwise resist condensation of viral genomes during encapsidation. Viruses typically accomplish this by sequestering positive charges in the form of divalent cations, such as Mg²⁺, or through polyvalent polyamine cations, such as spermine and spermidine or other small, basic cationic proteins that bind and condense nucleic acids with high affinity (7, 14, 19, 22). An example of the latter group is histones, which are abundant in the nucleus and high in arginine and lysine content, their primary role being to condense host chromosomal DNA and package it into nucleosomes (3). During virion assembly, histones are involved in condensation of viral genomes by several DNA viruses, including polyomaviruses, papovaviruses (8, 9, 20, 27), and herpes viruses (25), and by RNA retroviruses (31).Despite their ubiquity in the nucleus, histones do not appear to be involved in genome condensation and packaging of most viruses. For example, viruses as diverse as hepatitis B virus (HBV), baculoviruses, and other DNA viruses that replicate in the nucleus encode cationic proteins rich in arginine and lysine, a characteristic they share with histones and protamines. The latter are small, arginine-rich proteins that replace histones on chromosomal DNA during the late stages of spermatogenesis (1). Similar proteins have been found in viruses. The carboxy terminus of the HBV core antigen (16), for example, and the small P6.9 (6.9-kDa) proteins of baculoviruses (38, 39) are protaminelike virion components known to bind and condense viral DNA for encapsidation.Viruses of the family Ascoviridae are highly pathogenic to larvae of the lepidopteran family Noctuidae. The virions typically are large (130 to 150 nm by 200 to 400 nm) and have complex symmetry and organization consisting of an inner particle containing a protein/DNA core that after assembly is enveloped to form the virion (11, 12). Although the ultrastructure of ascoviruses is markedly different from those of baculoviruses, iridoviruses, and entomopoxviruses (33), all of these viruses have large, double-stranded DNA (dsDNA) genomes, typically >100 kbp, that must be condensed for packaging. However, whereas the protaminelike P6.9 protein of baculoviruses is known to be a key protein involved in condensing genomic DNA and packaging it into the virion, the proteins responsible for this function in the other DNA viruses that attack insects remain unknown. Sequence analysis of the Spodoptera frugiperda ascovirus 1a (SfAV1a) genome (4), the type species, revealed no genes coding for small, protaminelike peptides that could facilitate condensation and packaging of its large genome (157 kbp). Moreover, of 21 proteins revealed by our recent proteomic analysis of the SfAV1a virion (35a), no small, cellular histones or histonelike protamines were detected. The two most abundant proteins in the SfAV1a virion were the major capsid protein (MCP) and a protein with a mass of 64 kDa (ORF048 or P64) (4, 35a). As MCPs and core DNA-condensing and -packaging proteins generally occur in amounts proportionally greater than those of other structural proteins, the relative abundance of P64, and its high pI of 12.2, suggested that it might be a key protein involved in condensing and packaging the SfAV1a genome. In the present study, we characterized P64 and show that it is a novel protein containing two distinct domains with two distinct motifs. The amino-terminal portion contains four repeats of a well-conserved virus-specific two-cysteine adaptor motif (pfam08793.1) (18), whereas the carboxy-terminal portion contains 14 copies of a previously uncharacterized motif rich in arginine and serine residues (SPSQRRSTS[V/K][A/S]RR) and, to a lesser extent, several lysine residues. Through a combination of DNA-binding and gel shift assays, along with immunogold labeling electron microscopy, we demonstrate that P64 is a major virion protein in the SfAV1a DNA/protein core and may be involved in packaging this virus'' genome into the virion.     

The temporal and spatial expression pattern of myosin Va, Vb and VI in the mouse ovary

There are 16 classes of unconventional myosins. Class V myosins have been shown to be involved in transporting cargo to and from the cell periphery. Class VI myosins have also been shown to transport cargo from the cell periphery, although it seems that these proteins have many roles which include the mediation of cell migration and stereocillia stabilisation. With the requirement of myosin VI for Drosophila oogenesis, the localised expression of Myosin V in the developing egg chamber and recent mounting evidence which links myosin VI to the migration of human ovarian cancer cell lines, we wanted to investigate the expression pattern of these two myosin classes in the normal mouse ovary. Here we show that these myosins are expressed, localised and regulated within the oocyte and granulosa cells of the developing mouse follicle.     

The Product of the fimI gene is necessary for Escherichia coli type 1 pilus biosynthesis

Site-directed mutagenesis was employed to create lesions in fimI, a gene of uncertain function located in the chromosomal gene cluster (fim) involved in Escherichia coli type 1 pilus biosynthesis. Chromosomal fimI mutations produced a piliation-negative phenotype. Complementation analysis indicated that a fimI'-kan insertion mutation and a fimI frameshift mutation produced polarity-like effects not seen with an in-frame fimI deletion mutation. Minicell analysis associated fimI with a 16.4-kDa noncytoplasmic protein product (FimI). We conclude that FimI has a required role in normal pilus biosynthesis.     

The role of neurotrophin receptors in female germ-cell survival in mouse and human

During mammalian ovary formation, the production of ovarian follicles is accompanied by an enormous loss of germ cells. It is not known how this loss is regulated. We have investigated the role of the Trk tyrosine kinase receptors, primarily TrkB, in this process. The ovaries of TrkB-/- and TrkC-/- mice with a mixed (129Sv x C57BL/6) genetic background were examined shortly after birth. Around 50% of TrkB-/- mice had grossly abnormal ovaries that contained greatly reduced numbers of follicles. No defects were found in the ovaries of TrkC-/- mice. Congenic TrkB-/- mice were generated on 129Sv and C57BL/6 backgrounds: whereas the former had a mixed ovarian phenotype similar to that of the original colony of mice, the ovaries of all offspring of the C57BL/6 congenic line contained reduced numbers of follicles. RT-PCR showed that mRNA encoding TrkB and its two ligands, neurotrophin 4 (NT4) and brain-derived neurotrophic factor (BDNF), were present throughout the period of follicle formation in the mouse. In situ hybridisation showed that TrkB was expressed primarily in the germ cells before and after follicle formation. Mouse neonatal and fetal ovaries and human fetal ovaries were cultured in the presence of K252a, a potent inhibitor of all Trk receptors. In mice, K252a inhibited the survival of germ cells in newly formed (primordial) follicles. This effect was rescued by the addition of basic fibroblast growth factor (bFGF) to the culture medium. Combined addition of both BDNF and NT4 blocking antibodies lowered germ-cell survival, indicating that these TrkB ligands are required in this process. The results indicate that signalling through TrkB is an important component of the mechanism that regulates the early survival of female germ cells.     

Influence of supplemental high molecular weight pullulan or gamma-cyclodextrin on ileal and total tract nutrient digestibility, fecal characteristics, and microbial populations in the dog

This study was conducted to determine if supplemental pullulan and gamma-cyclodextrin affect canine nutrient digestibility, microbial populations, and fecal characteristics. Ileal cannulated dogs were fed a commercial diet, and treatments were administered daily in a 5 x 5 Latin square design: (i) no supplement; (ii) 2 g pullulan; (iii) 4 g pullulan; (iv) 2 g gamma-cyclodextrin; (v) 4 g gamma-cyclodextrin. Ileal and fecal samples were collected the last 4 d of each 14-d period. Increasing pullulan tended (p < 0.10) to linearly increase ileal bifidobacteria and lactobacilli and quadratically increase fecal lactobacilli. A similar response was noted in ileal bifidobacteria and lactobacilli with gamma-cyclodextrin. Gamma-Cyclodextrin resulted in a quadratic decrease (p < 0.05) in fecal Clostridium perfringens. Increasing pullulan linearly increased (p < 0.05) fecal score, while gamma-cyclodextrin resulted in a linear decrease (p < 0.05). Pullulan and gamma-cyclodextrin supplementation may have beneficial effects on the microbial ecology of dogs.     

Genetic characterization of Escherichia coli type 1 pilus adhesin mutants and identification of a novel binding phenotype

Five Escherichia coli type 1 pilus mutants that had point mutations in fimH, the gene encoding the type 1 pilus adhesin FimH, were characterized. FimH is a minor component of type 1 pili that is required for the pili to bind and agglutinate guinea pig erythrocytes in a mannose-inhibitable manner. Point mutations were located by DNA sequencing and deletion mapping. All mutations mapped within the signal sequence or in the first 28% of the predicted mature protein. All mutations were missense mutations except for one, a frameshift lesion that was predicted to cause the loss of approximately 60% of the mature FimH protein. Bacterial agglutination tests with polyclonal antiserum raised to a LacZ-FimH fusion protein failed to confirm that parental amounts of FimH cross-reacting material were expressed in four of the five mutants. The remaining mutant, a temperature-sensitive (ts) fimH mutant that agglutinated guinea pig erythrocytes after growth at 31 degrees C but not at 42 degrees C, reacted with antiserum at both temperatures in a manner similar to the parent. Consequently, this mutant was chosen for further study. Temperature shift experiments revealed that new FimH biosynthesis was required for the phenotypic change. Guinea pig erythrocyte and mouse macrophage binding experiments using the ts mutant grown at the restrictive and permissive temperatures revealed that whereas erythrocyte binding was reduced to a level comparable to that of a fimH insertion mutant at the restrictive temperature, mouse peritoneal macrophages were bound with parental efficiency at both the permissive and restrictive temperatures. Also, macrophage binding by the ts mutant was insensitive to mannose inhibition after growth at 42 degrees C but sensitive after growth at 31 degrees C. The ts mutant thus binds macrophages with one receptor specificity at 31 degrees C and another at 42 degrees C.     

Interfacial conditions between a press-fit acetabular cup and bone during daily activities: implications for achieving bone in-growth

Interfacial gaps and relative micromotions during activities are widely believed to restrict the boney in-growth process of non-cemented acetabular cups. Using finite element modeling of the cup-bone system, relative micromotions and interfacial gaps are calculated for walking slow, normal and fast and for climbing upstairs, downstairs and standing up from a chair. A 2mm press-fit is simulated and interfacial conditions in the immediate postoperative period (i.e. prior to boney in-growth) calculated between paired nodes covering the whole of the interface. In regions of 'safe' micromotions and 'allowable' gaps, boney in-growth is simulated by specifying zero relative displacement between nodal pairs. The modified model is then resubjected to the loads associated with climbing upstairs, which was shown to be the worst activity. Interfacial conditions are recalculated for subsequent iterations. The procedure is repeated until no further in-growth is predicted. The final pattern of in-growth calculated with the model compares reasonably well with histological evidence from explanted canine cups (Cha et al., 1998. Transactions of the Orthopaedic Research Society, 23, p. 373). Bridging between adjacent regions of in-growth is observed. Notably, in-growth occurs at most of the periphery but not in the polar region. The lack of polar in-growth is caused by the interfacial gap assumed to exist after cup implantation. It is suggested that increasing/decreasing hip-joint loads would have little effect on this lack of polar in-growth. However, excessive micromotions as a result of high hip-joint loads cause a lack of in-growth in the anterior region of the periphery in the model. Although such results were not found in the canine study, if relevant to the general human population, the avoidance of harsh weight-bearing activities may encourage complete peripheral in-growth but is speculated to do little for polar in-growth.     

Failure of founder transgenic male mice to transmit an attenuated HSV thymidine kinase transgene results from mosaicism and sperm competition

Previously we found that male mice carrying either of two attenuated herpes simplex virus thymidine kinase reporter transgenes displayed low level ectopic expression of the reporter gene in the testis and, although fertile, exhibited reduced fecundity. In contrast to males of later generations, many of the founder males failed to transmit the transgene to their progeny. This led to the suggestion that these fertile non-transmitting males are mosaic, with the sperm developing from the non-transgenic lineage outperforming those from the heterozygous transgenic lineage. Here we present the results of artificial insemination (AI) and in vitro fertilization (IVF) experiments designed to test this hypothesis. Albino CF(1) hybrid females were inseminated with mixtures of equal numbers of sperm from heterozygous transgenic (HT) males (equivalent to C57BL/6 x CBAF(2)) and CF(1) males. Similar mixed inseminations were carried out in parallel with sperm from non-transgenic (NT) siblings of the HT mice and 13-day fetuses were scored by eye color to determine their paternity. The pooled data from five experiments gave ratios of CF(1) to HT and CF(1) to NT offspring of 8.13 and 0.22 respectively, implying a calculated HT to NT ratio of 0.027. This indicates that, in competition with each other, the NT sperm would be almost 40 times more successful in fertilization than the HT sperm. Smaller differences were observed between HT and NT when AI was performed with unmixed sperm, consistent with the fertility of HT non-founder males. However, in five IVF experiments carried out with unmixed sperm, 142/212 oocytes exposed to NT sperm were activated and divided, while only 8/226 oocytes treated with HT sperm reached the two-cell stage. This confirms that HT sperm are defective and indicates that the IVF method employed amplified these deficiencies, which may have only a small effect upon natural reproduction when the HT sperm are not in competition with normal sperm.