Characterization of a retinoic acid responsive element isolated by whole genome PCR.

We have used whole PCR in an attempt to isolate novel retinoic acid (RA) responsive genes. We cloned several small genomic fragments from total human DNA containing putative retinoic acid responsive elements (RAREs) selected by direct binding to the retinoic acid receptor alpha (RAR alpha). We report here that an oligonucleotide containing a sequence from one of the cloned human DNA fragments, and referred to as alpha 1, functions as an authentic RARE. It is shown that both RAR alpha and RAR beta produced in Cos cells as well as in vitro translated RAR alpha bind directly and sequence-specifically to the alpha 1RARE. By mutational analysis it is demonstrated that the alpha 1RARE consists of an imperfect direct repeat of the estrogen- and thyroid hormone-related AGGTCA half-site motif separated by a 5 bp spacer. The orientation and spacing of the half-site repeats are shown to play a critical role in RAR recognition. When cloned upstream of a TK-Luc reporter, the alpha 1RARE is shown to confer responsiveness to RA in an orientation-independent fashion in F9 and CV-1 cells. The magnitude of the RA response mediated by the alpha 1RARE differed in these cell lines.

Heterogeneity of the polyribocytidilic acid tract in aphthovirus: changes in the size of the poly(C) of viruses recovered from persistently infected cattle.

A sample of aphthovirus type C3 strain Resende carrying two polyribocytidilic acid [poly(C)] tracts was cloned in tissue culture. One clone with a poly(C)-rich tract of about 145 nucleotides long (clone 3B) and another with a poly(C)-rich tract of about 230 nucleotides long (clone 12) and a mixture of both were injected intralingually into three steers. Samples from all three animals were recovered during the acute phase of the disease, from the blood and from the feet, and at various days after inoculation from the oesophageal-pharyngeal (OP) fluids. Analysis of the viral RNAs of the positive samples by means of RNase T1 maps on one- and two-dimensional gels showed (1) changes in the electrophoretic mobility of the poly(C)-rich tracts of viruses recovered from the OP fluids at various times after infection; (2) selection of virus populations with poly(C)-rich tracts of increased size; (3) later on, changes in the patterns of oligonucleotides of persistent viruses. These variations may lead to the production of new strains with altered biological properties that may contribute to the maintenance and spread of these viruses in the field.

Independent regulation of tumor necrosis factor and lymphotoxin production by human peripheral blood lymphocytes.

We present evidence that human peripheral blood lymphocytes, free of contaminating monocytes, rapidly produce high levels of tumor necrosis factor (TNF) when stimulated with phorbol diester and calcium ionophore, and lower but significant levels of TNF when stimulated with mitogens. These two types of inducers act preferentially on T cells, both CD4+ and CD8+. NK cells produce TNF only when stimulated with phorbol diester and calcium ionophore, and they do so at a much lower level than T cells. The procedures used in the purification of lymphocytes and the differential ability to respond to various inducers allow us to exclude that monocytes or basophils contaminating the lymphocyte preparation participate in the production of TNF. In particular, LPS, a potent inducer of TNF production from monocytes, is unable to induce significant levels of TNF in the lymphocyte preparations. The TNF produced by lymphocytes has antigenic, physicochemical, and biochemical characteristics identical to those of the TNF produced by myeloid cell lines or monocytes upon stimulation with LPS. LT is also produced by lymphocyte preparations. Production of TNF and LT proteins in response to the different inducers is paralleled by accumulation of cytoplasmic TNF and LT mRNA. Both at mRNA and at protein levels, stimulation of T lymphocytes with phorbol diester and calcium ionophore preferentially induces TNF, whereas mitogen stimulation preferentially induces LT. Our data suggest that the TNF and LT genes, two closely linked genes encoding two partially homologous proteins with almost identical biological functions, are independently regulated in lymphocytes.

RNA fingerprinting of South American prototype aphthovirus strains.

Aphthovirus strains used in South America for vaccine production or as reference for diagnostic purposes were analysed by RNA fingerprinting (RNase T1 maps, one- and two-dimensional gels). The results obtained constitute the basis for a data bank containing available information about the genome structure of strains of aphthovirus prevalent in this continent and can be used as an adjunct to serological and immunological information. These data are currently being used in South American countries to assess the genetic stability of strains during vaccine production; to establish possible vaccine origin of field outbreaks and to monitor the origin, behaviour and fate of new strains in the field.

Heterogeneity of the polyribocytidylic acid tract in aphthovirus: biochemical and biological studies of viruses carrying polyribocytidylic acid tracts of different lengths.

In this paper we report a study of a sample of foot-and-mouth disease virus carrying two polyribocytidylic acid [poly(C)] tracts of different lengths. By plaque purification in tissue culture, we isolated two populations of particles, one carrying the long poly(C) tract and the other carrying only the short homopolymer. The fingerprints of both viruses were indistinguishable from each other and from that of the virus present in the original sample, suggesting that the main difference between the two types of particles is limited to the poly(C) tracts of their genomic RNAs, to the flanking sequences of the poly(C) tract, or to both. In addition, some biological properties of these viruses are reported, such as stability upon serial passages in different cell lines, plaque size, and pathogenicity for cattle. The results indicate that the size of the poly(C) tract is not directly related to the virulence of these viruses. However, the size of the homopolymer could play a role in determining their efficiency of replication, and it appears that the particles with the short poly(C) tract might have some replicative advantage over those carrying the long one.