Characterization of a mouse interferon gene locus I. Isolation of a cluster of four alpha interferon genes.

A BALB/c mouse genomic library was screened with a murine interferon alpha 2 (MuIFN-alpha 2) cDNA coding region fragment. Eight clones were isolated which contain different mouse chromosomal segments related to the MuIFN-alpha 2 probe and a 28 kilobase (kb) region of mouse genomic DNA containing four different MuIFN-alpha genes (alpha 1, alpha 4, alpha 5 and alpha 6) was identified and characterized; an intergenic 1000 nucleotide long conserved sequence was found to be associated with three of these four alpha genes, indicating that this alpha-IFN gene cluster evolved through tandem duplications. Sequence analysis revealed the absence of a polyadenylation site in the 3' untranslated region of MuIFN-alpha 1, and showed that one of the genes (alpha 4) contains an internal deletion of 5 amino acids in the coding region.     

Synthesis of fusion and mature murine alpha interferons in Escherichia coli

Four murine interferon-alpha (MuIFN-alpha) genes (alpha 1, alpha 4, alpha 5, alpha 6) were previously identified and characterized. The coding regions of these IFN-alpha genes were inserted into bacterial expression vectors behind the lpp promoter under the control of the lac promoter-operator region, resulting in fusion peptides containing additional N-terminal amino acids (aa). Plasmids coding for the expression of mature IFN-alpha 1 and alpha 5 were also constructed using the same vector system, by inserting a 30-bp synthetic oligodeoxynucleotide, which contains a stop codon for the lpp gene, a ribosome-binding sequence and an ATG start codon for the IFN peptides. The amounts of IFN polypeptides synthesized in Escherichia coli were estimated in the maxi-cell system and their biological activities were measured on mouse and other mammalian cells. The yields of mature IFN produced in this vector were 2 to 4 X 10(6) units/liter; the antiviral activity of the majority of the MuIFNs on human and bovine cells was 100- to 1000-fold lower than on mouse cells. IFN-alpha 4, which contains an internal deletion of 5 aa, showed a lower antiviral activity than other MuIFNs on mouse cells.     

Structure and expression of a new murine interferon-alpha gene: MuIFN-alpha I9

A new murine alpha interferon gene, MuIFN-alpha I9, isolated from a BALB/c genomic clone, was characterized. It encodes a mature polypeptide of 167 amino acids (aa), presenting from 77 to 86% homology with the seven other MuIFN-alpha I aa sequences previously described. When compared to the latter, pre-IFN-alpha I9 has 13 distinctive aa, and, remarkably, ten of these occur in pairs. The coding region, fused to the SV40 early promoter and introduced into COS monkey cells, directed the transient secretion of an acid-stable functional IFN of 18-21 kDa. The production in this system reached levels of 300 000 units per 0.15 ml. A comparison of the aa sequence of different murine, rat, bovine, and human alpha and beta IFNs revealed certain common features allowing us to propose a putative secondary structure of the IFN proteins. A detailed analysis of results previously published by us and by others showed that the MuIFN-alpha I9 gene is, together with a least twelve other MuIFN-alpha I genes, located on chromosome 4.     

Differential antiproliferative activities of IFNs alpha, beta and gamma: kinetics of establishment of their antiproliferative effects and the rapid development of resistance to IFNs alpha and beta

Interferons have been recognized to have potent in vitro antiproliferative activities in mouse and human systems. To further investigate the kinetics of development of interferons' antiproliferative activities, mouse B-16 melanoma cells were treated with MuIFN-alpha, MuIFN-beta or MuIFN-gamma for various initial periods of time during an 8 day cloning assay. With MuIFN-alpha and MuIFN-beta treatments, maximal expression of antiproliferative activity was attained with 2 to 4 days of interferon treatment. In contrast, with MuIFN-gamma treatment, expression of antiproliferative activity increased with progressively longer periods of time of MuIFN-gamma treatment. These results suggested that B-16 melanoma cells were initially sensitive to all three of the interferons but rapidly became resistant to MuIFN-alpha and MuIFN-beta after 2 to 4 days of treatment. This suggestion was confirmed by cell growth kinetics experiments. The cells which were resistant to the antiproliferative activity of the MuIFN-alpha remained sensitive to the antiviral activity of MuIFN-alpha, suggesting that MuIFN-alpha and MuIFN-beta regulate their antiviral and antiproliferative responses via different mechanisms. The cells which were resistant to the antiproliferative activities of MuIFN-alpha and MuIFN-beta remained sensitive to MuIFN-gamma, suggesting that they were not generally resistant to antiproliferative effects. The cells which were resistant to the antiproliferative activities of the interferons gradually lost their resistance with a half-life of 11 days when they were cultured in the absence of interferons. The differential antiproliferative actions of alpha, beta and gamma interferons observed with murine B-16 melanoma were confirmed in the human system with G-361 melanoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chromosomal localization, expression pattern, and promoter analysis of the mouse gene encoding adipocyte-specific secretory protein Acrp30

Acrp30 is an abundantly expressed secretory protein exclusively synthesized in adipose tissue. Due to the dysregulation in various forms of obesity in humans and mice and its strong structural similarity to TNFalpha, it is currently under study as an important molecule involved in whole body energy homeostasis. Here we describe the sequence of mouse Acrp30 locus, define the intron/exon boundaries and map the gene to the telomere of mouse chromosome 16, syntenic to the human chromosomal locus 3q27. We demonstrate that alternative polyadenylation gives rise to two distinct mRNA species. We also show that Acrp30 expression is induced only at the late stages of mouse embryonic development. Finally, we have characterized the mouse Acrp30 promoter in tissue culture cells. We propose that Acrp30 promoter has the potential to drive strong adipocyte specific heterologous gene expression in transgenic mice.     

Overproduction of biologically-active human nerve growth factor in Escherichia coli.

A gene coding for human nerve growth factor (hNGF) was constructed for expression under control of the trp promoter in E. coli. The plasmid pTRSNGF contained a synthetic hNGF gene fused, in frame, to the region encoding the beta-lactamase signal peptide. The plasmid pTRLNGF contained the same coding sequence as hNGF attached downstream from the N-terminal fragment of the trp L gene. E. coli cells harboring pTRSNGF produced an amount of hNGF constituting 4% of the total cellular protein, and removed the beta-lactamase signal peptide. The mature protein hNGF was biologically active in the PC12h bioassay for neurite outgrowth. This biological activity was comparable to that of authentic mouse NGF. E. coli cells harboring pTRLNGF produced an amount of fusion protein hNGF constituting 25% of the total cellular protein. Although the fusion protein hNGF formed inclusion bodies in cells, dissolved fusion protein hNGF was active in neurite outgrowth from PC12h cells.     

Characterization of murine interferon-alpha 12 (MuIFN-alpha12): biological activities and gene expression

Interferon alpha (IFN-alpha) belongs to the type I interferon family and consists of multiple subtypes in many species. In the mouse, there are at least 14 IFN-alpha genes and 3 IFN-alpha pseudogenes, the most recently identified of which are murine interferon-alpha 12 (MuIFN-alpha12), MuIFN-alpha13 and MuIFN-alpha14. To further study the biological activities of MuIFN-alpha12, we have produced a recombinant MuIFN-alpha12 (rMuIFN-alpha12) protein using COS-1 cells. rMuIFN-alpha12 was found to inhibit the growth of murine myeloid leukemia JCS cells. Flow cytofluorometric analysis with propidium iodide staining showed that the growth inhibitory activity of rMuIFN-alpha12 may be caused by the induction of apoptosis. Flow cytofluorometric analysis also revealed that rMuIFN-alpha12 was able to up-regulate the expression of MHC-I on both JCS cells and primary macrophages. Functional studies indicated that a MuIFN-alpha12 transgene could induce an anti-viral state in L929 cells against Influenza A virus. Moreover, expression of MuIFN-alpha12 was not detectable by RT-PCR in untreated, Influenza A virus infected, polyI:polyC induced L929 cells, or in a wide range of normal murine tissues. Taken together, this data shows that MuIFN-alpha12 is a protein with all the biological traits of a type I IFN.     

Engineering Viral Promoters for Gene Transfer to Human Neuroblasts

1. The strength and activity of several viral promoters in human neuroblasts were evaluated in vitro. 2. Several luciferase reporter gene contructs under the control of different viral promoters (HIV-1 LTR, HTLV-I LTR, MMTV LTR, RSV LTR, CMV, SV40), in the presence or in the absence of the viral SV40 enhancer, were transfected into two well-established human neural cell lines, including one derived from human embryonic olfactory cells (B4) and one derived from an adrenal neuroblastoma (SH-SY-5Y). The epithelial cell line HeLa was used as a control.3. The enzymatic activity of luciferase was evaluated after normalization with an internal control. The results indicated that in the context of the reporter gene constructs, the CMV promoter alone was, overall, the most active in any tested cell line. However, addition of the SV40 enhancer to the CMV promoter abolished luciferase activity in SH-SY-5Y cells while significantly increasing luciferase expression in the CNS derived B4 fetal neuroblasts.4. The results suggest that gene therapeutic vectors aimed to promote enzymatic activity through gene transfer into undifferentiated human neural cells are feasible. However, since differences in promoter activity in neuroectodermal-derived cells are very relevant, gene construct variants should be considered to optimize the system.     

Effect of c-myc gene expression on early inducible reactions required for erythroid differentiation in vitro.

By employing cell fusion between two genetically marked mouse erythroleukemia (MEL) cells in which an artificially introduced c-myc gene had been placed under the control of human metallothionein promoter, we investigated the mechanism of the suppressive action of c-myc gene expression in erythroid differentiation. The results indicated that the expression of the c-myc gene blocked the induction of dimethyl sulfoxide-inducible activity, one of the two early activities required for triggering the differentiation.