Biomedical subjects
V Cunliffe
Publications and source records attributed to V Cunliffe.
Inhibition of Tat-mediated transactivation and HIV replication with Tat mutant and repressor domain fusion proteins.
Strategies to inhibit the spread of HIV infection consist of a number of specific molecular approaches. Since viral production is dependent upon Tat-mediated transactivation of the HIV promoter through the Tat activating region (TAR), tat antisense RNA, anti-tat ribozymes, TAR decoys and dominant negative Tat mutant proteins have been suggested as therapeutic inhibitors. We produced and tested several Tat mutant proteins, including a newly generated form Tat delta 58, for the ability to inhibit Tat-mediated transactivation and HIV production. In addition, we generated a new Tat fusion mutant between a C-terminus truncated form of Tat (Tat delta 53) and the Drosophila Engrailed (Eng) transcription repressor domain to test the hypothesis that transcriptional repression can be targeted to the HIV promoter. This fusion mutant was also examined for its capacity to block both Tat-mediated transactivation and HIV replication. We show that three mutants Tat delta 53. Tat delta 58 and Tat delta 53/Eng result in a transdominant phenotype inhibiting wild-type Tat-mediated transactivation, and that the inhibiting potential is increased by the presence of the entire basic domain or the fusion of a repressor domain. However, only the transdominant mutants Tat delta 58 and Tat delta 53/Eng significantly inhibit HIV-1 replication after infection of transfected T cell lines. These results demonstrate the potent inhibiting activity of Tat mutants on HIV replication, and suggest a synergistic effect of Tat transdominant mutant fusion with the Drosophila Engrailed transcription repressor domain.
The Xenopus Brachyury promoter is activated by FGF and low concentrations of activin and suppressed by high concentrations of activin and by paired-type homeodomain proteins.
The mesoderm of Xenopus laevis arises through an inductive interaction in which signals from the vegetal hemisphere of the embryo act on overlying equatorial cells. One candidate for an endogenous mesoderm-inducing factor is activin, a member of the TGFbeta superfamily. Activin is of particular interest because it induces different mesodermal cell types in a concentration-dependent manner, suggesting that it acts as a morphogen. These concentration-dependent effects are exemplified by the response of Xbra, expression of which is induced in ectodermal tissue by low concentrations of activin but not by high concentrations. Xbra therefore offers an excellent paradigm for studying the way in which a morphogen gradient is interpreted in vertebrate embryos. In this paper we examine the trancriptional regulation of Xbra2, a pseudoallele of Xbra that shows an identical response to activin. Our results indicate that 381 bp 5' of the Xbra2 transcription start site are sufficient to confer responsiveness both to FGF and, in a concentration-dependent manner, to activin. We present evidence that the suppression of Xbra expression at high concentrations of activin is mediated by paired-type homeobox genes such as goosecoid, Mix.1, and Xotx2.
Innovative approaches to gene therapy.
The past year has witnessed several advances in the development of targeted, cell-specific gene delivery systems of both viral and non-viral origin. Progress has been made in understanding the cellular mechanisms of nuclear import, and novel sequence-specific integrases have been developed that mediate insertion of DNA molecules into specific target sequences. Knowledge of the mechanisms by which herpes simplex virus and Epstein-Barr virus escape from immune surveillance has also advanced significantly; this may be exploited to reduce the immunogenicity of certain therapeutic gene products.
Patterning of the mesoderm in Xenopus: dose-dependent and synergistic effects of Brachyury and Pintallavis.
Widespread expression of the DNA-binding protein Brachyury in Xenopus animal caps causes ectopic mesoderm formation. In this paper, we first show that two types of mesoderm are induced by different concentrations of Brachyury. Animal pole explants from embryos injected with low doses of Xbra RNA differentiate into vesicles containing mesothelial smooth muscle and mesenchyme. At higher concentrations somitic muscle is formed. The transition from smooth muscle formation to that of somitic muscle occurs over a two-fold increase in Brachyury concentration. Brachyury is required for differentiation of notochord in mouse and fish embryos, but even the highest concentrations of Brachyury do not induce this tissue in Xenopus animal caps. Co-expression of Brachyury with the secreted glycoprotein noggin does cause notochord formation, but it is difficult to understand the molecular basis of this phenomenon without knowing more about the noggin signal transduction pathway. To overcome this difficulty, we have now tested mesoderm-specific transcription factors for the ability to synergize with Brachyury. We find that co-expression of Pintallavis, but not goosecoid, with Brachyury causes formation of dorsal mesoderm, including notochord. Furthermore, the effect of Pintallavis, like that of Brachyury, is dose-dependent: a two-fold increase in Pintallavis RNA causes a transition from ventral mesoderm formation to that of muscle, and a further two-fold increase induces notochord and neural tissue. These results suggest that Pintallavis cooperates with Brachyury to pattern the mesoderm in Xenopus.
Specification of mesodermal pattern in Xenopus laevis by interactions between Brachyury, noggin and Xwnt-8.
We demonstrate that the nuclear, sequence-specific DNA-binding protein Xbra causes dorsal mesodermal differentiation of animal cap ectoderm when co-expressed with the secreted proteins noggin and Xwnt-8. None of these molecules causes dorsal mesoderm formation when expressed alone. Co-expression of Xenopus Brachyury (Xbra) mRNA with noggin mRNA in animal caps specifies the main dorsal tissues, namely muscle, notochord and neural tissue. Co-expression of Xbra with Xwnt-8, in contrast, converts animal caps to muscle masses. We have previously shown that expression of Xbra alone in animal caps is sufficient to specify ventral mesoderm which expresses Xhox3 and low levels of muscle-specific actin. We now conclude that the putative transcription factor Xbra defines a cell state in the vertebrate embryo which can respond to diffusible dorsal signals such as noggin and Xwnt-8, resulting in dorsal mesodermal differentiation. In the absence of such dorsal signals Xbra causes ventral mesodermal differentiation. This state is only partially maintained after the mid-blastula transition as it permits the dorsal response to zygotically expressed noggin but it does not allow a dorsal response to zygotically expressed Xwnt-8, which elicits only ventral mesodermal differentiation.
Intercellular signalling in mesoderm formation during amphibian development.
The mesoderm of amphibian embryos arises through an inductive interaction in which a signal from the vegetal hemisphere of the blastula-stage embryo acts on overlying equatorial cells. Strong candidates for endogenous mesoderm-inducing signals include members of the fibroblast growth factor (FGF) and activin families. In this paper we show that cells form different mesodermal cell types in response to different concentrations of these factors, and that graded distributions of activin and FGF can, in principle, provide sufficient positional information to generate the body plan of the Xenopus embryo.
Ectopic mesoderm formation in Xenopus embryos caused by widespread expression of a Brachyury homologue.
The Brachyrury (T) gene is required cell-autonomously for mesoderm formation in the posterior of the mouse embryo, and both is complementary DNA sequence and expression pattern closely resemble those of a Xenopus homologue (Xbra), suggesting that these genes have an evolutionarily conserved function in vertebrate development. Strong expression of Xbra messenger RNA is found in the ring of involuting mesoderm during Xenopus gastrulation, and the expression of Xbra is an immediate-early response of animal pole blastomeres to mesoderm-inducing factors. To assess the role of Xbra in mesoderm formation, we increased its domain of expression in the embryo by microinjection of Xbra transcripts into the animal pole of Xenopus embryos at the one-cell stage. We show that expression of Xbra by cells of the early embryo is sufficient to direct their development into differentiated mesodermal tissues. At the molecular level this response shows a sharp threshold of sensitivity to the dose of Xbra RNA delivered, and we suggest that Xbra may act as a genetic switch initiating posterior mesodermal specification during embryogenesis.
A mouse zinc finger gene which is transiently expressed during spermatogenesis.
Zinc finger proteins are polypeptides with sequence-specific, nucleic acid-binding properties. Substantial evidence has established them as a class of trans-acting molecules with regulatory roles in cellular growth and differentiation. We have screened an 11.5 day post coitum urogenital ridge cDNA library with an oligonucleotide encoding a sequence conserved between a variety of zinc finger proteins. By cDNA cloning and sequencing we show that a novel mouse gene, Zfp-35, encodes a protein with a block of 18 zinc finger domains and an N-terminal region rich in acidic residues. The 2.4 kb mRNA encoding this polypeptide is selectively expressed in adult testis, by comparison with other organs. We have analysed Zfp-35 expression in whole testes of sex-reversed mice, whole testes of prepuberal XY animals, germ cell fractions from XY adult testes and by in situ hybridization to sections from adult XY testes. Our studies show that a considerable increase in expression is restricted to spermatocytes at the pachytene stage of meiotic prophase. These experiments suggest that Zfp-35 may act to control gene activity during this particular stage of spermatogenesis.
Genomic analysis of a mouse zinc finger gene, Zfp-35, that is up-regulated during spermatogenesis.
Zinc finger genes are a class of eukaryotic regulatory genes that encode sequence-specific nucleic acid-binding proteins. Members of this large gene family are required for growth and development in a wide range of organisms. We previously identified a mouse zinc finger gene, Zfp-35, that was up-regulated during spermatogenesis at the pachytene spermatocyte stage of development. We now describe the genomic organization of this gene, including its intron-exon structure, the sequence of its flanking regions, and its assignment to a region encompassing bands B3 to C of chromosome 18. The transcription unit has three exons. Intron 1 is within the 5' untranslated region and exon 3 contains the block of all 18 zinc fingers. These two features are common to a number of zinc finger genes. We also show that Zfp-35 is conserved in some placental mammals and that it is a member of a subfamily of related mammalian zinc finger genes.
The molecular genetics of human chromosome 6.
Chromosome 6 contains several clinically important markers as well as classical enzyme loci, proto-oncogenes, and a growing number of anonymous DNA restriction fragment length polymorphisms (RFLPs). It is also of unique interest because of the location of the major histocompatibility complex (MHC) on the short arm, at 6p21.3. The MHC is one of the most detailed areas of the human genetic map to date and many important diseases, some of a suspected autoimmune aetiology, are associated with it.