Molecular biology & medicine最新文献

Second generation therapeutic proteins are now being produced by in vitro mutagenesis of the relevant genes. Of some concern, however, is the possibility that these altered proteins will be immunogenic and the antibodies raised will also recognize the endogenous protein with undesirable consequences. We have designed a biological system to test these possibilities. In this model, transgenic mice produce and secrete human tissue plasminogen activator (h.tPA) to which these mice are immunologically tolerant. However, when challenged with a form in which a single amino acid has been substituted these mice will produce antibodies capable of recognizing h.tPA. These results indicate that there are immunological consequences connected with the design and administration of second generation therapeutic proteins.

The effects of polyoma (Py) oncogenes in transgenic mice are reviewed, with emphasis on an analysis of mice carrying individual Py early region genes linked to either the Py early region promoter or to the rat insulin II promoter. The perturbations documented in this series of animals varied with both the Py gene and the promoter. Py promoter-driven transgenes led to the development of pituitary tumors and vascular tumors in a gene-dependent manner, suggesting that the Py T antigens have different tissue specificities of transforming activity. The only pathology found in insulin promoter-driven transgenic mice was a beta cell tumor seen in insulin-promoted Py large T antigen-carrying mice. These results also suggest that the Py oncogenes have different tropisms relative to a specific cell type. Analysis of the Py oncogene-induced perturbations may provide both insights into the molecular events controlling tumorigenesis and access to rare or dispersed cell types for further analysis.

The mouse downless (dl) gene is a morphogenetic gene that plays a role in dermal-epidermal interaction and regulation of hair follicle induction during fetal development. We report here the identification of a transgenic mouse line with an insertional mutation in the dl gene. The genomic sequences flanking the transgenic insert have been cloned and used as hybridization probes to confirm that the mutant transgenic mice are homozygous for the transgenic insert and that the site of integration lies on mouse chromosome 10. Genomic probes that are close to or within the downless gene are now available and should permit the characterization of a gene that is involved in induction of a specific type of epithelial morphogenesis.

The soluble form of human CD4, an HIV receptor molecule first detected on the surface of T cells, binds glycoprotein gp120, a coat protein of human immunodeficiency virus, and has potential value for the treatment of AIDS. As a first step toward providing the necessary quantities of this protein at an affordable price we report here on the production of functional, soluble human CD4 in transgenic mice. In these animals, a regulatory region derived from a murine gene encoding the whey acidic protein directs synthesis of human CD4 protein to the mammary gland of lactating animals where it is secreted into milk.

Three strains of transgenic mice carrying the mouse metallothionein-I (MT) promoter fused to the human transthyretin (TTR) structural gene plus pUC plasmid sequences were investigated for expression of the fusion gene. Human TTR was inducible in the serum of at least two strains and the fusion gene mRNA was detected in several tissues of all the strains. The testis showed constitutive mRNA synthesis, while the intestine and some other tissues showed inducible expression. Unexpectedly, however, the fusion gene activity was grossly suppressed in the liver and kidney of all the strains. Available data suggest that this suppression results from the presence of the plasmid sequences. Analysis of tissue DNAs shows that the methylation status of the promoter sequences varies from strain to strain, depending on their chromosomal position, and that some CpG sites in the proximal portion of the promoter are not methylated at all in the liver and kidney of two strains. These findings suggest that the plasmid sequences suppress the MT promoter activity in some specific tissues by a mechanism that does not involve DNA methylation.

Most transgenic animals have been produced by directly injecting DNA into one of the embryo pronuclei in the period immediately following fertilization. Transgenic animals are produced when DNA becomes integrated into the chromosomes, in most cases the transgenic genotype is transmitted to progeny through the germ line. The characteristics of the foreign DNA include tandem arrangement of multiple copies of the input DNA, predominantly in direct rather than inverted orientation, and illegitimate recombination with the chromosome at the site of integration. Foreign DNA integrated into chromosomes of cultured cells has identical characteristics. We argue that these and other similarities between the integration of foreign DNA into the chromosomes of microinjected embryos on the one hand and on the other of transfected and microinjected cells, strongly suggest that the processes have the same basis. By considering mainly the literature relating to cell transfection and microinjection, we conclude that tandemly arranged concatemers of input foreign DNA are built up by a process of homologous but non-conservative recombination. End-joining and illegitimate recombination events characterize the integration of the concatemers into the chromosome, and also contribute to the formation of the concatemers. We also suggest that these superficially different processes are based on the same opportunistic repair-ligation mechanism; the frequencies of the different types of event reflect both the frequencies with which different sorts of association occur between DNA molecules and their relative stability.

Expression vectors encoding cDNAs for the human platelet-derived growth factor (PDGF) A-chain (pJKl) and murine dihydrofolate reductase (pTKDHFR) were cotransfected into dihydrofolate reductase-deficient Chinese hamster ovary cells. Methotrexate-induced coamplification of clones, expressing PDGF A-chain resulted in enhanced levels of A-chain-specific DNA, RNA and protein. A 30,500 Mr protein was immunoprecipitated with PDGF antisera from the conditioned media of metabolically labeled cells. Reducing conditions resolved the A-chain-specific protein into two polypeptides of 16,500 and 17,000 Mr, confirming the homodimeric nature of the recombinant A-chain protein. The recombinant PDGF A-chain produced constitutively by these amplified clones proved to be mitogenically active. The secretion of a recombinant PDGF A-chain into conditioned media may provide a continuous and abundant source of PDGF A.A dimers, normally produced by specific tissues in only minute quantities. Future purification of the recombinant homodimeric A-chain will allow the assessment of its ability to function in clinical applications such as wound healing.

We have reported that a liver growth factor isolated from plasma of partially hepatectomized rats is an albumin-bilirubin complex. In this paper, we characterize the liver growth factor purified from subjects with hepatitis (h-LGF). This factor increases synthesis of DNA in a dose-dependent manner both in vivo in mouse hepatocytes, with a dose of maximal stimulation of 150 ng of h-LGF/mouse, and in vitro in rat liver cell culture, with maximal effect at 7.5 to 10 ng of h-LGF/ml. In vivo, h-LGF increases the mitotic index of mouse hepatocytes, its action being organ-specific, acting on liver, but not on spleen, kidney, lung or brain. In vitro, h-LGF stimulates the uptake of 22Na+ by hepatocytes. In addition, we carried out a study comparing it with human serum albumin in terms of absorbance, fluorescence, circular dichroism spectra, amino acid composition, tryptic maps and antigenic determinants (Ouchterlony immunodiffusion). All these tests suggested that human serum albumin is a constituent of h-LGF. Moreover, when albumin isolated from humans without hepatic pathology is incubated with bilirubin, the albumin-bilirubin complex formed mimics the activity of the human liver growth factor with respect to stimulation of DNA synthesis and the effects on the mitotic index of mouse hepatocytes in vivo. We propose that this human liver growth factor is an albumin-bilirubin complex.

CpG dinucleotides have been implicated as mutational hotspots in genes that are subject to control mechanisms involving methylation. We have used the polymerase chain reaction to amplify exons 2 and 6 of the human antithrombin III gene and direct sequencing to identify the base replacement in 12 genetic variants. These occurred in individuals with a history of thromboembolic disease due to functional abnormalities of circulating antithrombin: ten had decreased heparin binding and activation, two had decreased inhibitory activity. The amino acid abnormality in ten out of 12 cases had arisen at a CpG dinucleotide; this confirms the CpG sequence as a "hotspot" in the antithrombin gene and explains the observed frequency of occurrence of the same variant antithrombins in diverse populations.