Receptor-mediated interference mechanism responsible for resistance to polytropic leukemia viruses in Mus castaneus

The Asian mouse Mus castaneus is resistant to infection by the polytropic mink cell focus-inducing (MCF) subgroup of murine leukemia viruses (MuLVs). Genetic crosses showed this recessive resistance to be governed by a single gene that maps at or near the gene encoding the polytropic viral receptor, Rmc1. To investigate this resistance, we mated M. castaneus with mice carrying the wild mouse Sxv variant of the Rmc1 receptor that allows infection by xenotropic as well as polytropic virus. Unlike other F1 hybrids of M. castaneus, these F1 mice were resistant to both xenotropic and polytropic classes of MuLVs. Analysis of backcrossed progeny of the F1 hybrids mated to Sxv mice indicates that resistance is due to inheritance of two M. castaneus genes. Cells from individual backcross mice were also examined for cell surface antigen by fluorescence-activated cell sorter analysis with monoclonal antibodies reactive with xenotropic or MCF virus env glycoproteins. A correlation was observed between virus resistance and antigen, suggesting that virus resistance is due to expression of endogenous viral envelope genes that interfere with infection by exogenous virus. Since the inbred strain Rmc1 receptor remains functional in the presence of these M. castaneus genes, and since M. castaneus contains multiple copies of xenotropic MuLV env genes, we suggest that these resistance genes control expression of xenotropic env glycoprotein that interferes with exogenous virus in cells containing the Sxv variant of Rmc1.     

Establishment and maintenance of a regionalized glycoprotein distribution during early mouse development

The regionalization of the cell membranes of the mouse embryo into apical and basolateral zones has been studied using antibodies to a pair of glycoproteins expressed during the two-cell to early blastocyst stage. These antigens are found on the outer, free surface and in the underlying cortical cytoplasm, but are not detectable at areas of cell contact. In the early blastocyst stage, antigen also appears at the free surfaces of cells bordering the blastocoel. Antigen regionalization is also reestablished after experimental manipulation and appears to be a direct consequence of cell contact. Thus, blastomeres examined 4 hr after dissociation from four- and eight-cell stage embryos express antigen in cortical areas underlying newly exposed surfaces and new sites of contact between embryos in multiple-embryo aggregates lose detectable antigen within 2 to 4 hr of the formation of the contacts. Microfilaments are involved in controlling the regional expression of these glycoproteins. Incubation of embryos from the two-cell stage in medium containing cytochalasin B interferes with antigen targeting, resulting in abnormal expression of the antigens both on the surface and in the cytoplasm of the embryos. Cytochalasin B treatment of later stage embryos results in an uneven distribution of the antigen in cortical cytoplasm and prevents the complete removal of antigen from new sites of cell contact in multiple-embryo aggregates. The presence of nocodozole, which inhibits the polymerization of microtubules, had no detectable effect on the expression of the antigens. Interference with the glycosylation of these proteins, by incubation of embryos in the presence of tunicamycin, did not alter the regionalized pattern of expression.     

Activators of protein kinase C up-regulate the cell surface expression of CD2 and CD5 T cell glycoproteins

Activation of human T cells through the CD3-T cell receptor complex caused an augmentation in the cell surface expression of CD2 and CD5 glycoproteins. Evidence that protein kinase C is involved in the up-regulatory mechanism of these cell surface molecules has been obtained by three different approaches: (a) the changes in antigen expression were observed with activators of protein kinase C such as phorbol esters but not with activators of kinases dependent on calcium/calmodulin or cAMP; (b) the overexpression of CD2 and CD5 is also observed in cells treated with 1,2-dioctanoyl-rac-glycerol, an analogue of the physiological activator of protein kinase C; and (c) 1-(5-isoquinolinyl)-2-methylpiperazine, an inhibitor of protein kinase C but not N-(2-guanidinoethyl)-5-isoquinolinesulfonamide dihydrochloride, an inhibitor of the cAMP-dependent kinase, impairs CD2 and CD5 up-regulation. These changes in cell surface antigen expression appear to be caused by the concomitant increase in the mRNA levels for CD2 and CD5. Phosphorylation studies of the CD2 and CD5 glycoproteins indicated that the overexpression of these molecules was not associated with a specific pattern of phosphorylation since it was observed independently of their hyperphosphorylated or nonphosphorylated state.     

DNA rearrangements and antigenic variation in Trypanosoma equiperdum: multiple expression-linked sites in independent isolates of trypanosomes expressing the same antigen.

African trypanosomes resist the immune response of their mammalian hosts by varying the surface glycoprotein which constitutes their antigenic identity. The molecular mechanism of this antigenic variation involves the successive activation of a series of genes which code for different variant surface glycoproteins (VSGs). We have studied the expression of two VSG genes (those of VSG-1 and VSG-28) in Trypanosoma equiperdum, and we report the following findings. (i) The expression of both VSG genes is associated with the duplication and transposition of corresponding basic copy genes. (ii) The duplicated transposed copy appears to be the expressed copy. (iii) Although there are multiple genes which cross-hybridize with the VSG-1 cDNA probe, only one of these appears to be used as a template for the expression-linked copy in four independent BoTat-1 clones. (iv) Analysis of the genomic environments of the expressed VSG-1 genes from each of four independently derived BoTat-1 trypanosome clones revealed that there are at least three different sites into which the expression-linked copy can be inserted.     

Novel N-glycosylation in eukaryotes: laminin contains the linkage unit beta-glucosylasparagine

The linkage unit to protein of N-linked carbohydrate in eukaryotic glycoproteins consists of N-acetylglucosamine, coupled to the amido nitrogen of asparagine. Additional N-glycosyl linkage units have been unequivocally proven to exist only in the cell surface glycoproteins of various bacteria. Based on immunological analyses, isolation and chemical characterization, we report that one of these units, namely glucose linked to asparagine, exists in the mammalian protein laminin, an extracellular basement membrane component. This finding and the occurrence of identical disaccharide structures in archaebacterial cell surface glycoproteins and mammalian basement membrane protein complexes points towards a conserved and distinct function of these extracellular structural elements. In addition, a method is described to uncover a masked epitope in fixed tissues by chemical O-deglycosylation. This has allowed to morphologically localize the antigen beta-Glc-Asn by immunofluorescence to the basement membranes of kidney glomeruli.     

Expression and function of the Drosophila gene runt in early stages of neural development.

The Drosophila gene runt was initially identified on the basis of its role during segmentation. Recent molecular and genetic studies have demonstrated that the runt gene encodes a novel nuclear protein whose developmental importance is not exclusive to segmentation. This report addresses the functional relevance of runt expression in the developmental pathway of neurogenesis. Antibodies against the runt protein reveal that it is expressed in a subset of neuroblasts, ganglion-mother cells and neurons. A subset of these neurons also co-express the segmentation gene even-skipped (eve). Using eve as a marker, we show that runt is required for the normal development of these neurons. A runt P-transposon that lacks neural cis-regulatory elements is used to show that these neurons require runt activity independent of its activity during segmentation. These results are confirmed using a temperature-sensitive runt allele. Further temperature-shift experiments indicate that the requirement for runt is during an early stage of neurogenesis. Based on its pattern of expression and its temporal requirements, runt is distinguished as one of the earliest acting genes involved in the generation of diverse cell fates in the developing Drosophila nervous system.     

Expression of engrailed during segmentation in grasshopper and crayfish

We have used a monoclonal antibody that recognizes engrailed proteins to compare the process of segmentation in grasshopper, crayfish, and Drosophila. Drosophila embryos rapidly generate metameres during an embryonic stage characterized by the absence of cell division. In contrast, many other arthropod embryos, such as those of more primitive insects and crustaceans, generate metameres gradually and sequentially, as cell proliferation causes caudal elongation. In all three organisms, the pattern of engrailed expression at the segmented germ band stage is similar, and the parasegments are the first metameres to form. Nevertheless, the way in which the engrailed pattern is generated differs and reflects the differences in how these organisms generate their metameres. These differences call into question what role homologues of the Drosophila pair-rule segmentation genes might play in other arthropods that generate metameres sequentially.     

Pax group III genes and the evolution of insect pair-rule patterning

Pair-rule genes were identified and named for their role in segmentation in embryos of the long germ insect Drosophila. Among short germ insects these genes exhibit variable expression patterns during segmentation and thus are likely to play divergent roles in this process. Understanding the details of this variation should shed light on the evolution of the genetic hierarchy responsible for segmentation in Drosophila and other insects. We have investigated the expression of homologs of the Drosophila Pax group III genes paired, gooseberry and gooseberry-neuro in short germ flour beetles and grasshoppers. During Drosophila embryogenesis, paired acts as one of several pair-rule genes that define the boundaries of future parasegments and segments, via the regulation of segment polarity genes such as gooseberry, which in turn regulates gooseberry-neuro, a gene expressed later in the developing nervous system. Using a crossreactive antibody, we show that the embryonic expression of Pax group III genes in both the flour beetle Tribolium and the grasshopper Schistocerca is remarkably similar to the pattern in Drosophila. We also show that two Pax group III genes, pairberry1 and pairberry2, are responsible for the observed protein pattern in grasshopper embryos. Both pairberry1 and pairberry2 are expressed in coincident stripes of a one-segment periodicity, in a manner reminiscent of Drosophila gooseberry and gooseberry-neuro. pairberry1, however, is also expressed in stripes of a two-segment periodicity before maturing into its segmental pattern. This early expression of pairberry1 is reminiscent of Drosophila paired and represents the first evidence for pair-rule patterning in short germ grasshoppers or any hemimetabolous insect.     

Assignment of gene(s) coding for antigen defined by monoclonal antibody 2B2

A mouse monoclonal antibody (2B2) recognizes an antigen which is present on most human peripheral blood leukocytes but is absent from most proliferating cells. The antibody precipitated two surface-labeled membrane glycopolypeptides with molecular weights of 86,000 and 145,000, and it was strongly mitogenic to normal human lymphocytes. Somatic cell hybrids have been used for assigning the genes coding for these membrane glycoproteins to human chromosome 21. The assignment was based on correlation of antigen expression on mouse-human T-lymphocyte hybrids with the presence of human chromosomes in the same hybrid clones.     

Arthropod-like expression patterns of engrailed and wingless in the annelid Platynereis dumerilii suggest a role in segment formation

The origin of animal segmentation, the periodic repetition of anatomical structures along the anteroposterior axis, is a long-standing issue that has been recently revived by comparative developmental genetics. In particular, a similar extensive morphological segmentation (or metamerism) is commonly recognized in annelids and arthropods. Mostly based on this supposedly homologous segmentation, these phyla have been united for a long time into the clade Articulata. However, recent phylogenetic analysis dismissed the Articulata and thus challenged the segmentation homology hypothesis. Here, we report the expression patterns of genes orthologous to the arthropod segmentation genes engrailed and wingless in the annelid Platynereis dumerilii. In Platynereis, engrailed and wingless are expressed in continuous ectodermal stripes on either side of the segmental boundary before, during, and after its formation; this expression pattern suggests that these genes are involved in segment formation. The striking similarities of engrailed and wingless expressions in Platynereis and arthropods may be due to evolutionary convergence or common heritage. In agreement with similarities in segment ontogeny and morphological organization in arthropods and annelids, we interpret our results as molecular evidence of a segmented ancestor of protostomes.     

Synchronised cycling gene oscillations in presomitic mesoderm cells require cell-cell contact

Segmentation of the vertebrate body axis is initiated early in development with the sequential formation of somites. Somitogenesis is temporally regulated by a molecular oscillator, the segmentation clock, which acts within presomitic mesoderm (PSM) cells to drive periodic expression of the cyclic genes. We have investigated the kinetics of the progression of cycling gene expression along the PSM. Here we show that c-hairy1 and c-hairy2 mRNA expression traverses the PSM in an entirely progressive manner and that both these genes and c-Lfng maintain a similar anterior limit of expression during each cycle. However, some differences are seen regarding both the onset of a new oscillation of these genes and the duration of their expression in the caudal PSM. We also investigated whether oscillating cyclic gene expression in the PSM is entirely cell autonomous. We find that while small PSM explants are still able to maintain their oscillation schedule, once they are dissociated, PSM cells are no longer able to maintain synchronous oscillations. The results imply that cell communication or a community effect is essential for the normal pattern of cyclic gene expression in these cells.     

The immune response to Moloney murine leukemia virus-induced tumors: induction of cytolytic T lymphocytes specific for both viral and tumor-associated antigens

The specificity of CTL generated against tumors induced by murine leukemia viruses (MuLV) has been reported to parallel the expression of two serologically defined tumor cell surface antigens--the cross-reactive FMR antigen expressed on the surface of tumors induced by Friend, Moloney, and Rauscher MuLV, and the Gross cell surface antigen (GCSA) expressed on tumors induced by AKV/Gross MuLV. We examined the specificity of CTL generated against MuLV-induced tumors and identified two distinct patterns of reactivity. The first follows the traditional pattern of FMR vs GCSA reactivity as assessed on a panel of established MuLV-induced lymphomas. However, CTL exhibiting this pattern of reactivity are incapable of lysing MuLV-infected fibroblasts. CTL exhibiting the second pattern of reactivity are capable of lysing MuLV-induced lymphomas as well as MuLV-infected fibroblasts. In addition, these CTL exhibit extensive cross-reactivity between lymphomas and fibroblasts infected by both groups of MuLV. Our results suggest that CTL exhibiting the traditional FMR vs GCSA pattern of reactivity are directed against a tumor-associated antigen and not against virus-encoded antigens, and that CTL directed against MuLV-encoded antigens demonstrate extensive cross-reactivity, including the ability to lyse AKV-infected cells.     

analysis of genomic rearrangements associated with two variable antigen genes of Trypanosoma brucei.

Some variable surface glycoprotein (VSG) genes of Trypanosoma brucei undergo duplication and transposition when they are expressed. We report here the cloning of cDNAs coding for two VSGs from the ILtar 1 repertoire. Analysis of the genomes of trypanosomes expressing these and other antigens shows that there is no additional copy of the sequences coding for eight VSG in expressing clones of trypanosomes, and reveals rearrangements analogous to those previously described for the gene for another VSG from this antigen repertoire. The data indicate that duplication does not accompany the expression of these VSG genes. Transposition to a specific expression site cannot be excluded, but would have to involve either a much larger segment of DNA, or movement to a region of much greater homology with the previous flanking sequences, than is observed for VSG genes that are duplicated when expressed. It is reasoned that the control of expression by coupled duplication and transposition is not sufficient to account for the selection of a single VSG gene for expression.     

Expression of expansin genes is correlated with growth in deepwater rice.

Expansins are a family of proteins that catalyze long-term extension of isolated cell walls. Previously, two expansin proteins have been isolated from internodes of deepwater rice, and three rice expansin genes, Os-EXP1, Os-EXP2, and Os-EXP3, have been identified. We report here on the identification of a fourth rice expansin gene, Os-EXP4, and on the expression pattern of the rice expansin gene family in deepwater rice. Rice expansin genes show organ-specific differential expression in the coleoptile, root, leaf, and internode. In these organs, there is increased expression of Os-EXP1, Os-EXP3, and Os-EXP4 in developmental regions where elongation occurs. This pattern of gene expression is also correlated with acid-induced in vitro cell wall extensibility. Submergence and treatment with gibberellin, both of which promote rapid internodal elongation, induced accumulation of Os-EXP4 mRNA before the rate of growth started to increase. Our results indicate that the expression of expansin genes in deepwater rice is differentially regulated by developmental, hormonal, and environmental signals and is correlated with cell elongation.     

Characterization of Rat CD4 T Cell Clones Specific for the Major Surface Glycoprotein of Pneumocystis carinii

ABSTRACT. Pneumocystis carinii are coated by abundant heterogenous major surface glycoproteins (MSGs), which facilitate interaction with the host. We have produced MSG-specific T-cell clones from the spleens of P. carinii -exposed Lewis rats and analyzed five for antigen specificity to native MSG and a recombinant form of MSG, cell surface markers, and cytokine profiles. All five of the clones were CD4+. All of the clones proliferated specifically to both the native MSG and the recombinant MSG only in the presence of antigen presenting cells demonstrating that the response is antigen/driven rather than mitogen/driven. All five of the clones secreted IL-2 and IFN-γ, although in differing amounts, implicating a Th l response. Only one of the clones produced any detectable IL-4. This is the first report of T cell clones responsive to a specific antigen of P. carinii , MSG. We conclude that the T cell clones will be helpful in mapping protective epitopes present in MSG and in functional studies of MSG.     

Stabilizing patterning in the Drosophila segment polarity network by selecting models in silico

The segmentation of Drosophila is a prime model to study spatial patterning during embryogenesis. The spatial expression of segment polarity genes results from a complex network of interacting proteins whose expression products are maintained after successful segmentation. This prompted us to investigate the stability and robustness of this process using a dynamical model for the segmentation network based on Boolean states. The model consists of intra-cellular as well as inter-cellular interactions between adjacent cells in one spatial dimension. We quantify the robustness of the dynamical segmentation process by a systematic analysis of mutations. Our starting point consists in a previous Boolean model for Drosophila segmentation. We define mathematically the notion of dynamical robustness and show that the proposed model exhibits limited robustness in gene expression under perturbations. We applied in silico evolution (mutation and selection) and discover two classes of modified gene networks that have a more robust spatial expression pattern. We verified that the enhanced robustness of the two new models is maintained in differential equations models. By comparing the predicted model with experiments on mutated flies, we then discuss the two types of enhanced models. Drosophila patterning can be explained by modelling the underlying network of interacting genes. Here we demonstrate that simple dynamical considerations and in silico evolution can enhance the model to robustly express the expected pattern, helping to elucidate the role of further interactions.     

Comparison of the effects of Sindbis virus and Sindbis virus replicons on host cell protein synthesis and cytopathogenicity in BHK cells.

Infection of BHK cells by Sindbis virus leads to rapid inhibition of host cell protein synthesis and cytopathic effects (CPE). We have been studying these events to determine whether the expression of a specific viral gene is required and, in the present study, have focused our attention on the role of the structural proteins--the capsid protein and the two membrane glycoproteins. We tested a variety of Sindbis viruses and Sindbis virus replicons (virus particles containing an RNA that is self-replicating but with some or all of the viral structural protein genes deleted) for their abilities to inhibit host cell protein synthesis and cause CPE in infected BHK cells. Our results show that shutoff of host cell protein synthesis occurred in infected BHK cells when no viral structural proteins were synthesized and also under conditions in which the level of the viral subgenomic RNA was too low to be detected. These results support the conclusion that the early steps in viral gene expression are the ones required for the inhibition of host cell protein synthesis in BHK cells. In contrast, the Sindbis viruses and Sindbis virus replicons were clearly distinguished by the time at which CPE became evident. Viruses that synthesized high levels of the two membrane glycoproteins on the surface of the infected cells caused a rapid (12 to 16 h postinfection) appearance of CPE, and those that did not synthesize the glycoprotein spikes showed delayed (30 to 40 h) CPE.