Position-independent expression of a human nerve growth factor-luciferase reporter gene cloned on a yeast artificial chromosome vector.

Two yeast artificial chromosomes containing the entire human nerve growth factor gene were isolated and mapped. By homologous recombination a luciferase gene was precisely engineered into the coding portion of the NGF gene and a neomycin selection marker was placed adjacent to one of the YAC telomeres. Expression of the YAC-based NGF reporter gene and a plasmid-based NGF reporter gene were compared with the regulation of endogenous mouse NGF protein in mouse L929 fibroblasts. In contrast to the plasmid-based reporter gene, expression and regulation of the YAC-based reporter gene was independent of the site of integration of the transgene. Basic fibroblast growth factor and okadaic acid stimulated expression of the YAC transgene, whereas transforming growth factor-beta and dexamethasone inhibited it. Although cyclic AMP strongly stimulated production of the endogenous mouse NGF, no effect was seen on the human NGF reporter genes. Downregulation of the secretion of endogenous mouse NGF already occurred at an EC50 of 1-2 nM dexamethasone, but downregulation of the expression of NGF reporter genes occurred only at EC50 of 10 nM. This higher concentration was also required for upregulation of luciferase genes driven by the dexamethasone-inducible promoter of the mouse mammary tumor virus in L929 fibroblasts.

The type I interferon system is locally activated in psoriatic lesions.

The expression of mRNAs encoding interferons (IFNs) and IFN-inducible proteins has been studied in psoriatic lesions and in noninvolved skin. The specific mRNAs have been detected by in situ hybridization using antisense RNAs. Signals for the expression of IFN-gamma mRNA have been found exclusively in cells of psoriatic lesions, and most likely represent a subpopulation of infiltrating leukocytes. Weak signals of IFN-alpha mRNA have been detected throughout the hyperkeratotic epidermis, although specific signals for IFN-beta mRNA expression were not detectable. The expression of two IFN-alpha-inducible gene products, namely the MxA protein and the 2'-5' oligoadenylate (2-5A) synthetase, have been studied as markers for the local activation of the IFN-alpha system. Expression of MxA mRNA and protein was observed in psoriatic keratinocytes, but not in normal appearing keratinocytes adjacent to the lesions. Similarly, 2-5A synthetase expression was markedly elevated in psoriatic keratinocytes. The results of the present study indicate that the IFN-alpha system is selectively activated in psoriatic lesions, although it remains silent in noninvolved skin. The implications of this finding are discussed within the boundaries of current understanding of the cytokine network.

Expression of TGF-beta s and TGF-beta type II receptor mRNAs in mouse folliculogenesis: stored maternal TGF-beta 2 message in oocytes.

The present in situ hybridisation study describes expression of TGF-beta 1, TGF-beta 2, TGF-beta 3, and TGF-beta type II receptor mRNA during follicular development, and the temporal pattern and abundance of TGF-beta 2 transcripts in early pre-implantation embryos. TGF-beta 1 hybridisation signals were most prominent in the outer granulosa cell layers of the post-antral follicles and in the corpus luteum, whereas strong expression of TGF-beta type II receptor mRNA was confined to thecal cells. Weak TGF-beta 3 mRNA expression was observed in all major cell types of pre- and post-antral follicles. Most notably, the very strong TGF-beta 2 hybridisation signals which were detected in developing oocytes declined rapidly following fertilization. Although still abundant in two-cell embryos, TGF-beta 2 hybridisation signals were barely detectable in four- and eight-cell embryos. These findings, and the presence of specific sequence motifs in the 3' untranslated region of TGF-beta 2 mRNAs, suggest that TGF-beta 2 transcripts are stored as maternal messages.

In situ hybridization analysis of cytokine, proto-oncogene and tumour suppressor gene expression in psoriasis.

The purpose of this study was to investigate and to compare, by in situ hybridization, gene expression of IL-1 beta, IL-8, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-alpha, p53 and c-myc in lesions and in non-involved skin of patients with psoriasis. All lesional skin biopsies showed overexpression of IL-1 beta, IL-8 TGF-alpha mRNAs. IL-1 beta hybridization signals were strong in a small number of cells localized predominantly in the dermal papillae and in the suprapapillary epidermis. Overexpression of TGF-alpha was observed in all suprabasal keratinocytes, whereas strongly elevated IL-8 mRNA expression was found to be restricted to clusters of suprabasal keratinocytes. TGF-beta 3, p53 and c-myc transcripts were clearly detected in the epidermis of all biopsies, although expression levels were comparable in lesional and non-lesional skin.

Crystallization and preliminary X-ray analysis of recombinant human transforming growth factor beta 2.

Recombinant human transforming growth factor beta 2 (TGF-beta 2) was cloned and expressed in E. coli. The protein was isolated from inclusion bodies, renatured and purified to a single component as judged by reversed-phase HPLC. The recombinant TGF-beta 2 was shown to have a biological activity equal to that of native TGF-beta 2 in a fibroblast migration assay. Pure, active recombinant TGF-beta 2 has been crystallized from polyethylene glycol 400. The trigonal crystals of spacegroup P3(1)21 or P3(2)21 have unit cell dimensions of a=b=60.6 A, c=75.2 A and diffract beyond 2.0 A.

Wound healing in aged animals--effects of locally applied transforming growth factor beta 2 in different model systems.

Local application of a growth factor which could stimulate cell turnover, extracellular matrix synthesis and blood vessel formation in the skin should improve and accelerate wound healing processes which are often impaired in old age. We demonstrate the effects of TGF-beta 2 in promoting wound repair in old animals where normal healing responses are shown to be naturally slower. The potential use of TGF-beta s for the treatment of wound injuries, including chronic non-healing ulcers in the elderly, is discussed.

Localization of transforming growth factor-beta 1, -beta 2 and -beta 3 gene expression in bovine mammary gland.

We have studied the expression of transforming growth factor (TGF)-beta 1, -beta 2, and -beta 3 in the non-lactating and lactating bovine mammary gland by in situ hybridization. All three isoforms were expressed in the lobuloalveolar framework of the non-lactating and lactating gland although marked differences were apparent in their spatial distribution. TGF-beta 1 was expressed predominantly by the epithelial cells of the lobules although expression was also observed in the intralobular stroma cells lining the epithelium. In contrast, TGF-beta 2 expression was only observed in the epithelial cells. TGF-beta 3 transcripts were expressed at the highest levels and were observed in almost all cells of the lobule. No TGF-beta signals were found in the interlobular regions of the mammary gland. The possible regulatory functions of these molecules in development of the mammary gland and on differentiation processes in the neonate are discussed.

Differential expression of TGF beta 1, beta 2 and beta 3 genes during mouse embryogenesis.

We have examined by Northern analysis and in situ hybridisation the expression of TGF beta 1, beta 2 and beta 3 during mouse embryogenesis. TGF beta 1 is expressed predominantly in the mesodermal components of the embryo e.g. the hematopoietic cells of both fetal liver and the hemopoietic islands of the yolk sac, the mesenchymal tissues of several internal organs and in ossifying bone tissues. The strongest TGF beta 2 signals were found in early facial mesenchyme and in some endodermal and ectodermal epithelial cell layers e.g., lung and cochlea epithelia. TGF beta 3 was strongest in prevertebral tissue, in some mesothelia and in lung epithelia. All three isoforms were expressed in bone tissues but showed distinct patterns of expression both spatially and temporally. In the root sheath of the whisker follicle, TGF beta 1, beta 2 and beta 3 were expressed simultaneously. We discuss the implication of these results in regard to known regulatory elements of the TGF beta genes and their receptors.

Selective cloning of B cell hybridoma-specific rearranged immunoglobulin gene loci using the polymerase chain reaction.

The use of conventional DNA cloning procedures to obtain productively rearranged Ig genes from B cell hybridomas for structure/function analysis of immunoglobulins is tedious and time-consuming. Here we describe a procedure based on PCR which permits rapid, selective isolation of DNA segments containing individual hybridoma-specific Ig gene rearrangements. The method, an adaptation of the so-called 'inverted PCR' technique (IPCR), can be applied most efficiently to specific genes where a preliminary restriction map is available from Southern blot analysis of the hybridoma genomic DNA. To achieve amplification of a given rearranged Ig locus, small amounts of total hybridoma DNA are digested to completion with a chosen restriction endonuclease and the fragments circularised by DNA ligase. Cleavage of the DNA circles using a second restriction enzyme, chosen specifically to cut 3' to a rearranged V-(D)-J exon, leads to linear DNA segments where the rearranged gene is now flanked by segments of known nucleotide sequence derived originally from the 3' region of the Ig H or L chain gene locus. This permits the selection of oligonucleotides that provide convergent primers for specific amplification of DNA segments containing the required gene rearrangement. Amplified DNA fragments can be cloned and rapidly characterised by sequence analysis.

Cloning and sequence analyses of cDNAs for interferon- and virus-induced human Mx proteins reveal that they contain putative guanine nucleotide-binding sites: functional study of the corresponding gene promoter.

The human protein p78 is induced and accumulated in cells treated with type I interferon or with some viruses. It is the human homolog of the mouse Mx protein involved in resistance to influenza virus. A full-length cDNA clone encoding the human p78 protein was cloned and sequenced. It contained an open reading frame of 662 amino acids, corresponding to a polypeptide with a predicted molecular weight of 75,500, in good agreement with the Mr of 78,000 determined on sodium dodecyl sulfate gels for the purified natural p78 protein. The cloned gene was expressed in vitro and corresponded in size, pI, antigenic determinant(s), and NH2 terminus sequence to the natural p78 protein. A second cDNA was cloned which encoded a 633-amino-acid protein sharing 63% homology with human p78. This p78-related protein was translated in reticulocyte lysates where it shared an antigenic determinant(s) with p78. A putative 5' regulatory region of 83 base pairs contained within the gene promoter region upstream of the presumed p78 mRNA cap site conferred human alpha interferon (IFN-alpha) inducibility to the cat reporter gene. The p78 protein accumulated to high levels in cells treated with IFN-alpha. In contrast, the p78-related protein was not expressed at detectable levels. The rate of decay of p78 levels in diploid cells after a 24-h treatment with IFN-alpha was much slower than the rate of decay of the antiviral state against influenza A virus and vesicular stomatitis virus, suggesting that the p78 protein is probably not involved in an antiviral mechanism. Furthermore, we showed that these proteins, as well as the homologous mouse Mx protein, possess three consensus elements in proper spacing, characteristic of GTP-binding proteins.

A precise map of splice junctions in the mRNAs of minute virus of mice, an autonomous parvovirus.

We have determined the exact splicing patterns of the mRNAs of the minute virus of mice by a combination of cDNA sequencing and S1 nuclease protection analysis. There are four virus-specific mRNA species, each coding for one of the four polypeptides identified by in vitro translation. The R1 mRNA comprises sequences from nucleotide approximately 200 to 2281 and from 2378 to approximately 4800 and codes for the NS1 protein. The R2 mRNA is derived from nucleotides approximately 200 to 515, 1991 to 2281, and 2378 to approximately 4800 and codes for the NS2 protein. Between nucleotides 1991 and 2281, the coding sequence for NS2 overlaps that of NS1, but in a different reading frame. R3 covers nucleotides approximately 2007 to 2281 and 2378 to approximately 4800 and codes for VP2. The fourth species, R3', differs from R3 by using an alternative splice donor and acceptor in the region around 47 map units (nucleotide 2400); it extends from nucleotide approximately 2007 to 2317 and from 2400 to approximately 4800 and almost certainly codes for VP1. The R2 transcript is unusual in that the intron that was removed from it (nucleotides 516 to 1990) starts with GC rather than the canonical GU. With the exception of the splice acceptor at position 2378, which is found only in rodent parvoviruses, the splice junctions are highly conserved among autonomous parvoviruses. These results show that minute virus of mice, like other small DNA viruses, uses multiple strategies to compress the coding information for several viral proteins into a short (5,104 nucleotide) genome.

Cloning of eukaryotic protein synthesis initiation factor genes: isolation and characterization of cDNA clones encoding factor eIF-4A.

Monoclonal antibodies directed against rabbit reticulocyte protein synthesis initiation factor 4A (eIF-4A) were used to isolate mouse cDNA clones expressing eIF-4A protein sequences in E. coli. The identity of cDNA clones encoding eIF-4A sequences was confirmed by hybrid-selected translation and peptide mapping of the translation product. Analysis of the mRNA coding for eIF-4A from mouse liver and HeLa cells by Northern hybridization revealed two discrete mRNA species of approximately 2000 and 1600 nucleotides in length. The existence of two mRNAs in mouse and HeLa cells encoding eIF-4A was confirmed by cDNA sequencing.

DNA sequence comparison between two tissue-specific variants of the autonomous parvovirus, minute virus of mice.

We have determined the complete nucleotide sequence of the DNA of the immunosuppressive variant of the parvovirus minute virus of mice (MVMi) and compared it to the published sequence (12) of the fibroblast-specific strain (MVMp). We have found 175 differences between the two viruses, most of which affect single nucleotides. Despite these differences, the genomic organization of MVMp and MVMi is identical. There are 29 amino-acid changes between the putative viral gene products of MVMi and MVMp, 16 of which are conservative. We discuss the possibility that the differential tissue-specificity of the two variants is linked to differences within the non-transcribed region near the 5' end of the viral genomes.

Canine parvovirus: relationship to wild-type and vaccine strains of feline panleukopenia virus and mink enteritis virus.

Canine parvovirus (CPV), feline panleukopenia virus (FPLV) and mink enteritis virus (MEV) were compared serologically, by determination of their host range in cell cultures, as well as by restriction enzyme analysis. Maps of the virus genomes were established using seven different restriction enzymes cutting at a total of 56 sites. MEV and FPLV gave maps which were identical except for one restriction site. The map of CPV is closely related to those of FPLV/MEV since their DNAs share about 80% of the restriction sites tested. However, CPV is clearly distinct from FPLV/MEV since either eight (German isolate) or nine (Belgian, Swiss and American isolates) restriction sites are different. The DNAs of six vaccine strains of FPLV and MEV were also analysed. They gave maps which closely resembled those of the respective wild-type strains. CPV and FPLV/MEV also differed with respect to antigenicity, as well as to host range in cell cultures.

A mammalian DNA polymerase alpha holoenzyme functioning on defined in vivo-like templates.

In analogy to the Escherichia coli replicative DNA polymerase III we define two forms of DNA polymerase alpha: the core enzyme and the holoenzyme. The core enzyme is not able to elongate efficiently primed single-stranded DNA templates, in contrast to the holoenzyme which functions well on in vivo-like template. Using these criteria, we have identified and partially purified DNA polymerase alpha holoenzyme from calf thymus and have compared it to the corresponding homogeneous DNA polymerase alpha (defined as the core enzyme) from the same tissue. The holoenzyme is able to use single-stranded parvoviral DNA and M13 DNA with a single RNA primer as template. The core enzyme, on the other hand, although active on DNAs treated with deoxyribonuclease to create random gaps, is unable to act on these two long, single-stranded DNAs. E. coli DNA polymerase III holoenzyme also copies the two in vivo-like templates, while the core enzyme is virtually inactive. The homologous single-stranded DNA-binding proteins from calf thymus and from E. coli stimulate the respective holoenzymes and inhibit the core enzymes. These results suggest a cooperation between a DNA polymerase holoenzyme and its homologous single-stranded DNA-binding protein. The prokaryotic and the mammalian holoenzyme behave similarly in several chromatographic systems.

Characterization of an immunosuppressive parvovirus related to the minute virus of mice.

We have characterized an immunosuppressive parvovirus related to the minute virus of mice (MVM). The parvovirus, MVM(i), grew efficiently on the murine lymphoma cell line EL-4 and not on the A-9 strain of L-cells which is a host for the prototype MVM. MVM(i) was immunosuppressive for allogeneic mixed leukocyte cultures, inhibiting the generation of cytolytic T lymphocytes. MVM had no effect on mixed leukocyte cultures. MVM and MVM(i) particles were similar in buoyant density, sedimentation rate, appearance in the electron microscope, and polypeptide composition. We present restriction enzyme maps of the DNAs of MVM and MVM(i) which show that they are closely related. Out of 109 restriction endonuclease cleavage sites (representing together about 10% of the nucleotide sequence), 86 sites were shared by MVM and MVM(i), whereas 22 sites were absent from one of the two viruses. MVM(i) DNA had an apparent deletion of about 60 nucleotides relative to MVM, located near the 5' terminus of viral DNA.

Comparison of canine parvovirus with mink enteritis virus by restriction site mapping.

The genomes of canine parvovirus and mink enteritis virus were compared by restriction enzyme analysis of their replicative-form DNAs. Of 79 mapped sites, 68, or 86%, were found to be common for both types of DNA, indicating that canine parvovirus and mink enteritis virus are closely related viruses. Whether they evolved from a common precursor or whether canine parvovirus is derived from mink enteritis virus, however, cannot be deduced from our present data.