Regulating gene expression in transgenic animals.

Regardless of the field of application, the raison d'etre of transgenic animals is to study gene regulation and function. With increasing frequency, mammalian genes are being isolated with no concomitant knowledge of their function. The human genome mapping initiative will undoubtedly produce a cornucopia of such genes. While the merit of taking a transgenic route to study genes of unknown function is axiomatic, the choices of strategies for gene regulation in vivo may not be fully appreciated. This review will address two main points: first, the targeted and regulated expression of genes, and second, the structural and functional ablation of genes.     

T4 DNA replication and viral gene expression

The normal dependence of “late” T4 gene expression on concurrent viral DNA replication is circumvented in cells infected with a triple mutant in which viral DNA polymerase, DNA ligase, and the exonuclease functions of genes 46 or 47 are defective. Acrylamide gel electrophoresis of labeled proteins from infected cells has made possible an extension of the analysis of replication-uncoupled T4 protein synthesis. We find a number of late T4 proteins synthesized: the products of genes 34, 37, 18, 23 and 24. Processing of the gene 23 product, normally headassembly dependent, occurs, but with considerably diminished efficiency compared to wild-type infection. Late T4 protein synthesis in replication-uncoupled infection retains a requirement for T4 gene 33 and gene 55 function. Finally, a number of “early” T4 gene products, normally shut off late in wildtype infection, continue to be synthesized late in replication-uncoupled infection, concurrently with the late proteins.     

The first cycle of DNA replication in mouse embryogenesis studied by microinjections of 3H-thymidine into the cytoplasm of fertilized ova

H-thymidine was injected into cytoplasm of the eggs taken at different intervals after fertilization and the eggs were fixed immediately thereafter. DNA synthesis was shown to begin in pronuclei when they are still in the marginal zones of cytoplasm, immediately after their formation. S-phase lasts 5-6 h in every pronucleus and is terminated at 1-2 h before the first cleavage division when the pronuclei are closely approached and located in the center of cytoplasm. At the end of S-phase late replicating heterochromatic regions are distinctly localized near the nuclear envelope and in pronuclei. Male and female pronuclei display asynchrony in the course of S-phase and differences in 3H-thymidine incorporation into chromatin. Structural features of the first cell cycle in mouse embryogenesis are discussed.     

Global effects of DNA replication and DNA replication origin activity on eukaryotic gene expression

This report provides a global view of how gene expression is affected by DNA replication. We analyzed synchronized cultures of Saccharomyces cerevisiae under conditions that prevent DNA replication initiation without delaying cell cycle progression. We use a higher‐order singular value decomposition to integrate the global mRNA expression measured in the multiple time courses, detect and remove experimental artifacts and identify significant combinations of patterns of expression variation across the genes, time points and conditions. We find that, first, ～88% of the global mRNA expression is independent of DNA replication. Second, the requirement of DNA replication for efficient histone gene expression is independent of conditions that elicit DNA damage checkpoint responses. Third, origin licensing decreases the expression of genes with origins near their 3′ ends, revealing that downstream origins can regulate the expression of upstream genes. This confirms previous predictions from mathematical modeling of a global causal coordination between DNA replication origin activity and mRNA expression, and shows that mathematical modeling of DNA microarray data can be used to correctly predict previously unknown biological modes of regulation.     

Introduction of DNA sequences of Rous sarcoma virus into Drosophila and mouse genomes by microinjections into ova

The paper covers experimental results of introducing exogenic genetic material, namely DNA sequences of the Rous sarcoma virus, by microinjections in mice zygotes and Drosophila early embryos. In a number of cases integration of viral DNA into genomes of these organisms was detected. Blot-hybridizations analysis of cell DNA proved that the inserted viral sequences undergo rearrangements in the course of integration.     

The methods to generate transgenic animals and to control transgene expression

Transgenic animals have been used for years to study gene function and to create models for the study of human diseases. This approach has become still more justified after the complete sequencing of several genomes. Transgenic animals are ready to become industrial bioreactors for the preparation of pharmaceuticals in milk and probably in the future in egg white. Improvement of animal production by transgenesis is still in infancy.Despite its intensive use, animal transgenesis is still suffering from technical limitations. The generation of transgenics has recently become easier or possible for different species thanks to the use of transposons or retrovirus, to incubation of sperm which DNA followed by fertilization by intracellular sperm injection or not and to the use of the cloning technique using somatic cells in which genes have been added or inactivated. The Cre-LoxP system is more and more used to withdraw a given sequence from the genome or to target the integration of a foreign DNA. The tetracycline system has been improved and can more and more frequently be used to obtain faithful expression of transgenes. Several tools: RNA forming a triple helix with DNA, antisense RNA including double strand RNA inducing RNA interference and ribozymes, and also expression of proteins having a negative transdominant effect, are tentatively being improved to inhibit specifically the expression of host or viral genes.All these techniques are expected to offer experimenters new and more precise models to study gene function even in large animals. Improvement of breeding by transgenesis has become more plausible including through the precise allele replacement in farm animals.     

Modulation of gene expression in transgenic mouse hearts overexpressing calsequestrin

Calsequestrin (CSQ) is the major Ca2+ binding protein of the cardiac sarcoplasmic reticulum (SR). Transgenic mice overexpressing CSQ at the age of 7 weeks exhibit concentric cardiac hypertrophy, and by 13 weeks the condition progresses to dilated cardiomyopathy. The present study used a differential display analysis to identify genes whose expressions are modulated in the CSQ-overexpressing mouse hearts to provide information on the mechanism of transition from concentric cardiac hypertrophy to failure. Cardiac ankyrin repeat protein (CARP), glutathione peroxidase (Gpx1), and genes which participate in the formation of extracellular matrix including decorin, TSC-36, Magp2, Osf2, and SPARC are upregulated in CSQ mouse hearts at 7 and 13 weeks of age compared to those of non-transgenic littermates. In addition, two novel genes without sequence similarities to any known genes are upregulated in CSQ-overexpressing mouse hearts. Several genes are downregulated at 13 weeks, including SR Ca2+-ATPase (SERCA2) and adenine nucleotide translocase 1 (Ant1) genes. Further, a functionally yet unknown gene (NM_026586) previously identified in the mouse wolffian duct is dramatically downregulated in CSQ mice with dilated hearts. Thus, CARP, Gpx1, and genes encoding extracellular matrix proteins may participate in the development of cardiac hypertrophy and fibrosis, and changes in SERCA2, Ant1, and NM_026586 mRNA expression may be involved in transition from concentric to dilated cardiac hypertrophy.     

Relationship of eukaryotic DNA replication to committed gene expression: general theory for gene control.

The historic arguments for the participation of eukaryotic DNA replication in the control of gene expression are reconsidered along with more recent evidence. An earlier view in which gene commitment was achieved with stable chromatin structures which required DNA replication to reset expression potential (D. D. Brown, Cell 37:359-365, 1984) is further considered. The participation of nonspecific stable repressor of gene activity (histones and other chromatin proteins), as previously proposed, is reexamined. The possible function of positive trans-acting factors is now further developed by considering evidence from DNA virus models. It is proposed that these positive factors act to control the initiation of replicon-specific DNA synthesis in the S phase (early or late replication timing). Stable chromatin assembles during replication into potentially active (early S) or inactive (late S) states with prevailing trans-acting factors (early) or repressing factors (late) and may asymmetrically commit daughter templates. This suggests logical schemes for programming differentiation based on replicons and trans-acting initiators. This proposal requires that DNA replication precede major changes in gene commitment. Prior evidence against a role for DNA replication during terminal differentiation is reexamined along with other results from terminal differentiation of lower eukaryotes. This leads to a proposal that DNA replication may yet underlie terminal gene commitment, but that for it to do so there must exist two distinct modes of replication control. In one mode (mitotic replication) replicon initiation is tightly linked to the cell cycle, whereas the other mode (terminal replication) initiation is not cell cycle restricted, is replicon specific, and can lead to a terminally differentiated state. Aberrant control of mitotic and terminal modes of DNA replication may underlie the transformed state. Implications of a replicon basis for chromatin structure-function and the evolution of metazoan organisms are considered.