Molecular biology & medicine最新文献

When used in an in vitro transcription assay, the promoter of a cloned alpha 2u globulin gene is much more active in liver than in spleen nuclear extracts. Promoter deletion experiments suggest that both positive and negative regulatory mechanisms may be involved in the differential in vitro transcription from the alpha 2u globulin promoter in these two nuclear extracts. Interestingly, removal of promoter elements upstream from position -74 results in a significant increase of in vitro transcription in spleen but not in liver nuclear extracts, and thus reduces the difference in transcription observed with longer alpha 2u promoters in these two extracts. Deletion of additional nucleotides to position -43 strongly reduces the in vitro transcription efficiency of the promoter in extracts from both tissues. None of the examined promoters containing between 3000 and 22 nucleotides of 5' flanking regions are differentially transcribed in liver nuclear extracts from either male or female rats. Thus, in contrast to cell-type specificity, sex-specificity could not be observed in our in vitro transcription experiments. DNase I protection experiments with crude nuclear extracts and partially or highly purified nuclear proteins suggests the presence of six recognition sites for DNA-binding factors between the TATA element and position -210. Some of these factors could be identified as proteins that also bind to elements within the albumin gene promoter.

The specific functions expressed by differentiated cells are extinguished when these cells are crossed with somatic cells of another histotype or with cells of the same differentiation that fail to express these functions: in rat hepatoma x mouse fibroblast hybrids, tyrosine aminotransferase (TAT) and phosphoenolypyruvate carboxykinase (PEPCK) activities are extinguished as they are in hybrids between the same rat hepatoma cell line and mouse hepatoma cells that do not express these two enzymes. The locus Tse-1 (tissue-specific extinguisher) on mouse chromosome 11 is responsible for the extinction of TAT and PEPCK in rat hepatoma x mouse fibroblast hybrids and loss of mouse chromosome 11 leads to re-expression of these two enzymes. We report here an analysis of rat hepatoma x mouse hepatoma hybrids that demonstrates that loss of mouse chromosome 11 is not necessary for re-expression of TAT and PEPCK. In view of the facts that Tse-1 is active in mouse hepatoma cells that do not express TAT and PEPCK and that the presence of only one active Tse-1 locus is sufficient to extinguish these functions in 2s rat hepatoma cells, we conclude that re-expression of TAT and PEPCK in rat hepatoma x mouse hepatoma hybrids is due to the epistatic action of tissue-specific trans-acting activators that override the Tse-1 effect.

The envelope of human immunodeficiency virus (HIV) is an essential building block of the virus and it plays a major role in its life-cycle, particularly during the early stages of infection. It very likely determines, at least in part, the host range and tissue specificity of HIV, participates in pathogenic processes mediated by the virus and can itself be immunosuppressive. Because of its strategic location on the outer surface of the virion and the infected cell, it also represents an optimal (although not the only) target for immune attack and thus a prime candidate for development of vaccine and therapeutic strategies. Efforts to better understand its structural, functional and antigenic properties will thus be well worthwhile. Some of its principal features are reviewed herein and its role in vaccine strategies is discussed.

Two pathogenic human retroviruses have been isolated and shown to cause diseases characterized by malignant proliferation of T-cells. Human T-cell leukemia virus type I (HTLV-I) is the virus etiologic agent of adult T-cell leukemia, and human T-cell leukemia virus type II (HTLV-II) has been rarely associated with some forms of leukemia related to hairy-cell leukemia. Understanding the pathogenesis of these retroviruses requires elucidating the mechanism by which HTLV immortalizes cells. Two hypothetical modes of HTLV-induced transformation are discussed in this review. At the cell surface, HTLV particles via as yet unknown receptors have mitogenic effects on T-cell growth. Once HTLV productively infects the cell, it can initiate molecular changes as well. The HTLV genome encodes a viral regulatory protein, Tax, which not only activates HTLV gene expression, but may also induce inappropriate expression of cellular genes involved in cell proliferation. Models are proposed for how these events mediated by HTLV may contribute to T-cell transformation and ultimately, leukemia.

The simian virus 40 (SV40) tumor or T antigen synthesized in transformed or infected cells is highly immunogenic, inducing both antibody and cytotoxic T lymphocyte (CTL) responses. In the C57BL/6 (H-2b) strain of mice the CTL response is directed to discrete sites on T antigen. To date, five CTL recognition sites have been identified using CTL clones, deletion mutants and overlapping synthetic peptides. The CTL sites I, II and III are clustered in the amino-terminal one-third of T antigen, whereas sites IV and V are located in the carboxyl one-third. Using synthetic peptides, the site I has been tentatively assigned to residues 205 to 215 of T antigen and sites II and III map to residues 220 to 233. Site V maps to amino acids 489 to 503. The location of site IV remains undefined but probably falls between amino acids 368 and 511. The CTL sites I, II, III and V are H-2Db-restricted, whereas site IV is H-2Kb-restricted. CTL sites II and III can be distinguished using H-2Db class I mutants which present the same peptide differentially to CTL clones specific for sites II and III. The multiplicity of CTL sites on SV40 T antigen contributes to the overall immunosurveillance in the host against SV40 carcinogenesis. In the event of a loss of a particular site due to mutation or deletion, the remaining CTL sites continue to provide an effective target for CTL-mediated surveillance. Similar events may also contribute toward controlling papovavirus infections in humans.

Enveloped animal viruses enter their host cells by a process of membrane fusion. This fusion can occur at the cell plasma membrane or within the endocytic vacuolar system, depending on the characteristics of the virus fusion protein. Examples of both pathways of viral entry are detailed in this review. Semliki Forest virus (SFV) is presented as a well-studied prototype of those viruses which use endocytic uptake in order to infect cells. Fusion of endocytosed SFV is specifically triggered by the acidic pH present within the endocytic pathway, which causes specific conformational changes in the SFV spike protein. While the overall features of endocytic uptake are similar for all viruses which use this pathway, the mechanism by which the viruses then cause fusion appears to differ significantly between them. The best understood fusion mechanism is that of influenza virus, for which sequences involved in pH-dependent fusion can be correlated with the crystallographic structure of the spike protein. In contrast to these pH-dependent virus systems, the entry of human immunodeficiency virus (HIV) into cells occurs by a pH-independent fusion mechanism probably involving fusion at the plasma membrane. The data to date on HIV fusion, endocytosis and entry are summarized as an example of this pathway.

Human papillomaviruses (HPVs) have been detected in a wide range of proliferative lesions of squamous epithelium. A number of HPV types are associated with lesions of the genital tract. Some types (HPV types 6 and 11) are detected frequently in benign genital warts (condylomata acuminata); other types (HPV types 31, 33, 42 and others) are associated with dysplastic lesions of the uterine cervix; and certain HPV types (HPV types 16 and 18) are found in a high proportion of squamous cell carcinomas of the cervix and vulva. However, all of these HPV types have been detected also in normal epithelium. To date, investigators have relied primarily upon the detection of viral DNAs in clinical specimens as evidence of HPV infections. Such assays cannot determine whether past infections with HPVs have occurred which have subsequently resolved. Latent infections and current infections might also evade detection because of sampling errors or because of suboptimal sensitivity of DNA detection methods. Efforts to develop HPV serological assays have been hampered by the lack of appropriate viral antigens, since HPVs cannot be propagated in tissue culture and virions are not abundant in infected human tissues. Using HPV-encoded proteins expressed in Escherichia coli, we developed assays to measure human antibodies that react with HPV proteins. Human antibodies to late gene products (L1 and L2) of genital-type HPVs were more prevalent than antibodies to early gene products. However, approximately 15% of sera contained antibodies that reacted with the HPV16 E7-encoded protein, a gene product that has been implicated in HPV16-mediated cellular transformation. The human antibodies appeared to be type or "serotype" specific, because the antibodies did not cross-react with homologous proteins encoded by other HPV types. Antibodies to proteins encoded by HPV types 6 or 11 were detected in approximately 70% of adults, while antibodies to proteins encoded by HPV type 16 were found in approximately 50% of adults. Antibody prevalence was not associated with measures of sexual activity. There was also no significant difference between the prevalence of antibodies to HPV types 6 or 16 proteins in children when compared to the antibody prevalence in sexually active adults. These results suggest that infections by genital HPV types are widespread and frequently cause clinically inapparent infections. The viruses have a broad tropism for mucosal epithelium and are likely to be acquired by other modes, as well as by sexual transmission.

We have two goals in this review: the first is to relate what has been learned about DNA replication from the study primarily of herpes simplex virus type 1 (HSV-1); the second is to note briefly facets of this virus's mode of DNA replication that might serve as points of intervention for novel chemotherapeutic approaches in order to deal with primary and recurrent herpesvirus infections in man and animals. For the first goal we shall both summarize what has been learned and attempt to identify directions that may be pursued in order to further our understanding of DNA replication by herpesviruses. For the second goal we shall propose two schemes for the screening for drugs that might interfere uniquely with the DNA replication of this family of viruses.

For many bacterial species, entry into mammalian cells is an important step toward establishing an infectious disease. Genetic and molecular techniques have revealed many important features of the entry process. As an example of this approach, the enteric pathogen Yersinia pseudotuberculosis has been used as a model system for bacterial penetration. This analysis has uncovered at least three different pathways for entry of the microorganism into cultured mammalian cells. These pathways differ in regards to their tissue specificities as well as the regulatory signals that control their expression. One of these pathways, promoted by the Y. pseudotuberculosis outer membrane protein invasin, has been studied in detail. This single factor is sufficient to promote entry of inert particles by binding multiple integrin receptors during cellular uptake. The significance of multiple pathways for entry as well as the binding of multiple receptors is discussed.

Among a series of 44 transgenic families established after microinjection into fertilized eggs of a plasmid DNA where the structural gene for the large T antigen of polyoma virus is located downstream from the viral early promoter-enhancer region, one family with a hereditary neurological disorder was observed. At about three weeks of age, these animals developed a syndrome of constant tremor with recurrent seizures. Histological and ultra-structural examination revealed extensive dysmyelination in the white matter of the brain stem, cerebellum and spinal cord, as well as of peripheral nerves. This phenotype is reminiscent of that of the mouse "twitcher" (twi) mutant and of the human hereditary leukodystrophies. Expression of the viral sequences, assayed by Northern analysis and immunolabeling of T antigen, occurred predominantly in cells of the central nervous system. Integration of the transgene was mapped by in situ hybridization on metaphasic plaques in region B-C1 of chromosome 12 (where the twi locus was previously localized). Long-term cultures of cells with neural characteristics could be established readily from the brain of the transgenic mice.