Purification of a new restriction endonuclease, StyI, from Escherichia coli carrying the hsd+ miniplasmid

A new restriction endonuclease, StyI, free of contaminating nuclease activities, has been isolated from Escherichia coli carrying the hsd+ miniplasmid of Salmonella typhi origin. In the presence of 10 mM Mg2+, it recognizes and cleaves a hexanucleotide sequence of 5'-C decreases C(AT)(AT)GG. The advantages of the StyI endonuclease include its stability, high yield (more than 2 X 10(3) units/g of wet cells), easy handling of producer cells, and the ability to recognize new sequences, CCAAGG and CCTTGG.     

DNA-mediated genetic transformation of mouse embryos and bone marrow--a review

In recent years, new gene transfer systems have been developed which allow molecularly cloned genetic material to be introduced into whole organisms. These systems include the microinjection of DNA into mammalian embryos, transfection of DNA into mouse bone marrow cells, and the infection of early embryos with retroviruses. Exogenous DNA appears to integrate randomly into the host genome. The production of transgenic mice by injection of DNA into mouse embryos has rapidly gained importance as an experimental tool for the study of gene regulation during development. Through this technique, recombinant molecules of any type can be introduced into one-celled embryos, and thus can be used to study development from its earliest stages. DNA sequences have been shown to integrate and transmit through the germ line to subsequent generations as mendelian traits. Transgenic mice carrying various gene constructs have been successfully exploited for the elucidation of factors which determine tissue specificity of gene expression as well as the level of gene control. Phenotypic changes related to expression of foreign genes have also been observed. This experimental approach thus promises to rapidly solve many of the heretofore most challenging problems in developmental genetics. Insertion of foreign genes has also made possible the creation of insertional mutants which manifest themselves most frequently as recessives. Such mutations can be readily studied at the molecular level by using the transferred material as a probe for recovery of the affected host sequence from genomic libraries. Many of these same problems have been addressed by introducing retroviral DNA into mouse embryos. Here, the sequences used for transfer have been limited to retroviral genes, but nonetheless these experiments have been profitably exploited for studies both of gene regulation and mutagenesis. Gene transfer systems are being developed allowing the experimenter to transfer DNA into bone marrow cells of mice, after which the recipient cells can be reintroduced into lethally irradiated histocompatible animals. This system has the advantage that selection can be applied during the gene transfer process such that the expression of the foreign material is assured. In addition, these experiments have created a model system for production of animals carrying a subpopulation of cells which is highly resistant to a toxic agent. This system has the potential for therapeutic application to man.     

Circular dichroism of native whole SV40 chromatin.

Circular dichroism properties of SV40 virions, isolated minichromosomes from virions, and SV40 Form I (supercoiled) DNA were studied in a buffer of low ionic strength. The isolated minichromosomes are compact as judged by sedimentation and electron microscopy. The molar ellipticity at 284 nm of the virion, which may be regarded as a minichromosome in its native state, is about 1500 deg cm²/dmol phosphate; this value is in the same range as that reported for core particles (1300–2000) isolated from different sources. When the viral capsid is removed, there is a small increase in the molar ellipticity to about 2000. However, both of these values are much lower than that found for SV40 supercoiled DNA (about 8200). The results strongly suggest that the linker DNA of the native whole chromatin contributes in a similar fashion to the circular dichroic ellipticity as the core DNA.     

Antibodies reacting to mimotopes of Simian virus 40 large T antigen,the viral oncoprotein,in sera from children

Recent data indicate that the Simian virus 40 (SV40) infection appears to be transmitted in humans independently from early SV40-contaminated antipolio vaccines. Serum antibodies against SV40 large T antigen (Tag) were analyzed in children/adolescents and young adults. To investigate antibodies reacting to SV40 Tag antigens, serum samples ( n = 812) from children and young adults were analyzed by indirect ELISAs using specific SV40 Tag mimotopes. Mimotopes were synthetic peptides corresponding to SV40 Tag epitopes. In sera ( n = 412) from healthy children up to 17 years old, IgG antibodies against SV40 Tag mimotopes reached an overall prevalence of 15%. IgM antibodies against SV40 Tag were detected in sera of children 6–8 months old confirming and extending the knowledge that SV40 seroconversion occurs early in life. In children/adolescents affected by different diseases ( n = 180) SV40 Tag had a prevalence of 18%, being the difference no significant compared to healthy subjects ( n = 220; 16%) of the same age. Our immunological data indicate that SV40 circulates in children and young adults, both in healthy conditions and affected by distinct diseases. The IgM detection in sera from healthy children suggests that the SV40 infection/seroconversion occurs early in life (>6 months). Our immunological data support the hypothesis that SV40, or a closely related still unknown polyomavirus, infects humans. The SV40 seroprevalence is lower than common polyomaviruses, such as BKPyV and JCPyV, and other new human polyomaviruses. In addition, our immunological surveillance indicates a lack of association between different diseases, considered herein, and SV40.     

Inducible DNA-loop formation blocks transcriptional activation by an SV40 enhancer

It is well established that gene expression in eukaryotes is controlled by sequence-dependent binding of trans-acting proteins to regulatory elements like promoters, enhancers or silencers. A less well understood level of gene regulation is governed by the various structural and functional states of chromatin, which have been ascribed to changes in covalent modification of core histone proteins. And, much on how topological domains in the genome take part in establishing and maintaining distinct gene expression patterns is still unknown. Here we present a set of regulatory proteins that allow to reversibly alter the DNA structure in vivo and in vitro by adding low molecular weight effectors that control their oligomerization and DNA binding. Using this approach, we completely regulate the activity of an SV40 enhancer in HeLa cells by reversible loop formation to topologically separate it from the promoter. This result establishes a new mechanism for DNA-structure-dependent gene regulation in vivo and provides evidence supporting the structural model of insulator function.     

Differential expression of genes coding for EGF-like factors and ADAMTS1 following gonadotropin stimulation in normal and transformed human granulosa cells

We have demonstrated previously that the synthesis of epiregulin and amphiregulin, of the EGF-like growth factor family, is stimulated by luteinizing hormone in human follicular (granulosa) cells obtained from in vitro fertilization program. In the present work, we demonstrate that H89, a PKA inhibitor, attenuated the expression of these growth factors both in the mRNA and the protein levels, suggesting PKA involvement in this signaling pathway. SV40-transformed human granulosa cells showed higher basal levels of epiregulin and amphiregulin than normal cells, which were still elevated following cAMP stimulation by Forskolin. Cleavage by a disintegrin and metalloproteinases (ADAMs) is essential for activation of these growth factors, allowing their interaction with EGF receptor. Expression of ADAMTS1 and ADAM12 was downregulated by cAMP in normal, but not in SV40-transformed cells, suggesting that in normal cells epiregulin and amphiregulin activity is downregulated by a feedback mechanism that may be lost in SV40-transformed cells and their loss of downregulation may be involved in the development of ovarian tumors.