Heterologous Assay System Research Articles

A plant homologue to mammalian brain 14-3-3 protein and protein kinase C inhibitor

We have isolated cDNA clones of Spinacea oleracea L. and Oenothera hookeri of 930 and 1017 base pairs, respectively. The open reading frame deduced from the Oenothera sequence codes for a protein of a calculated molecular mass of 29 200. The primary amino acid sequence exhibits a very high degree (88%) of homology to the 14-3-3 protein from bovine brain, and protein kinase C inhibitor from sheep brain. Subsequently the plant protein was partially purified from leaf extract. The partially purified plant protein inhibited protein kinase C from sheep brain in a heterologous assay system. The active fraction consisted of 5–6 different polypeptides of similar molecular size. One of these proteins crossreacted with a peptide-specific antibody against protein kinase C inhibitor protein from sheep brain.

Identification and characterization of intragenic sequences which repress human immunodeficiency virus structural gene expression

Examination of the life cycle of the human immunodeficiency virus (HIV) has shown that multiple levels of regulation exist, including some which require the virus-encoded Rev protein. In the absence of Rev, mRNAs encoding the structural proteins remain untranslated, a phenomenon which appears, in part, to be caused by nuclear entrapment of these RNA species. To examine the basis for repression of structural gene mRNA expression, a heterologous assay system was utilized to determine whether regions present within gag and pol contain elements capable of suppressing gene expression when present in cis. Both genes were found to contain cis-acting repressor sequences (CRS) that block gene expression when present within the 3' untranslated portion of a heterologous gene transcript. The element within pol was found to have the strongest repressive effect. While Rev alone was unable to reverse the repression observed with the pol sequence, addition of the env Rev-responsive element (RRE) in cis and Rev in trans did cause reversal of inhibition. Deletion mutagenesis defined a 260-bp element within the 3' portion of pol that contains a potent CRS which functions when present in the sense orientation. The corresponding region in HIV-2 pol was found to contain a functionally similar CRS element. To examine the mechanism of repression, the effects of the CRS elements on both the abundance and subcellular distribution of the mRNAs were examined. Neither was dramatically altered when examined in the context of a heterologous reporter (chloramphenicol acetyltransferase) mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibin: clinical physiology and cancer diagnosis

Information on the physiology of circulating inhibin in man has to date been derived almost exclusively from measurements in peripheral blood, using a heterologous radio imrnuno assay (RIA) system (McLachlan et al. 1986a, 1987a). Although it appeared initially that this assay was highly specific for inhibin, it has been recognized more recently that inhibin-related proteins, present in follicular fluid (Robertson et al. 1989; Sugino et al. 1989; Robertson et al. 1990) and in peripheral blood (Schneyer et al. 1990) lack biological activity but cross react strongly in the RIA. Measurements obtained using this RIA must therefore be interpreted with caution (Robertson 1990). Although inhibin gene expression has been demonstrated in a variety of tissues (Meunier et al. 1988), inhibin levels are generally undetectable following castration and the menopause; this indicates that circulating levels are the result of gonadal production. Studies of circulating immunoreactive inhibin (irhh), particularly in the female, are consistent with the notion that this gonadal glyco-protein hormone is an important component of the hypothalamo-pituitary-ovarian axis and is involved in the feedback regulation of follicle stimulating hormone (FSH).

Complete mutagenesis of the HIV-1 protease.

Retroviruses encode a protease which needs to be active for the production of infectious virions. A disabling mutation in the protease results in the production of non-infectious virus particles and examination of proteins from these mutant virions reveals unprocessed Gag and Gag-Pol precursor proteins, the substrates of the viral protease. Each amino acid of the HIV-1 protease was individually mutated using a simple mutagenesis procedure which is capable of introducing and identifying missense mutations in each residue of a protein. Phenotypic screening of these mutants in a heterologous assay system reveals three regions within the protease where multiple consecutive amino-acid residues are sensitive to mutation. These results show that random mutagenesis can be used to identify functionally important regions within a protein. Mutants with conditional phenotypes have also been identified within this collection.

Multiple cis-active elements in the long control region of bovine papillomavirus type 1 (BPV-1).

A 1.0 kb region of the BPV-1 genome (the long control region, LCR), contains controls for transcription and the origin of replication. Transcription directed by the LCR is activated by the viral encoded E2 protein. To define the essential cis acting elements that are required to control transcription we have constructed a series of deletions throughout the LCR. We have identified three important domains in the LCR, two of which respond to E2. We have analysed the ability of small subcloned regions of the E2 responsive domains to act as enhancers in a heterologous assay system. This has led to the identification of five independent E2 responsive elements. We have shown that a fragment of only 38 base pairs is sufficient to respond to activation by E2. We also present evidence to suggest two types of E2 responsiveness that result in strong or weak activation. Strong response is correlated with the presence of the sequence 5'-ACCG/TNNNC/TCGGTGC-3' whereas weak response is correlated with the presence of a related sequence 5'-ACC(N)6GGT-3'. The contribution of these multiple elements to viral transcription is discussed.

Different profiles of isoelectric avian luteinizing hormone components in biological activity and immunoreactivity.

Chicken LH components from the glycoprotein fraction of the anterior pituitary glands have been separated to isoelectric homogeneity by means of isoelectric focusing, and investigated for their biological activities in vitro. The activities of these components were measured with LH receptor binding, cyclic AMP accumulation and testosterone production in rat Leydig cells at equal doses expressed as immunoreactivity of IRC-2 (Gunma). All the components were active in the heterologous assay systems, although the relative potency expressed as the ratios of biological activity to immunoreactivity (B:I) differed significantly among the components. Component I, the amount of which is the largest (40-50%), with the most alkaline isoelectric point (pI) showed the highest B:I ratio, and the B:I ratio decreased with decreasing pI in the same way as in rat LH components (Wakabayashi, 1980; Hattori et al., 1983). Therefore, pituitary LH from photostimulated male quail, where the relative amount of component I was increased, was estimated to have higher B:I ratios than that from short-day (SD) treated male quail. Furthermore, the differences in activities among the components obtained by the three assays were in the following order: testosterone production greater than cyclic AMP accumulation greater than receptor binding, suggesting that the hormonal actions of components with higher B:I ratios (I, II and III) are efficiently amplified in the biological response to the final step. In the incubation of pituitary glands with hypothalamic extracts, component I in the pituitary glands from the long-day (LD) treated group was mostly decreased after the incubation, while all the components were decreased in parallel in the SD-treated group. The results suggest that the LH component releasable to GnRH changes in endocrine status though component I with the highest B:I ratio is relatively releasable in both SD- and LD-treated groups.

Studies of circulating parathyroid hormone in man using a homologous amino-terminal specific immunoradiometric assay.

Circulating immunoreactive parathyroid hormone (PTH) was measured by a homologous, amino-terminal, specific, immunoradiometric assay in man. In forty-two healthy subjects the concentrations ranged between < 40 pg/ml and 120 pg/ml. No hormone could be detected in the sera of eleven patients with hypoparathyroidism, but the concentrations were clearly elevated in six patients with pseudohypoparathyroidism (range 190-1120 pg/ml). In thirty-five patients with primary hyperparathyroidism the mean (+/- SEM) concentration was 283.4 +/- 42.4 pg/ml (range 100-1350 pg/ml). A significant positive correlation was demonstrated between immunoassayable hormone and serum calcium concentrations in these patients. In nine patients PTH concentrations were measured before and after parathyroidectomy. In all of them they were elevated pre-operatively but fell to the normal range after parathyroidectomy. The disappearance of exogenously administered synthetic human PTH (1-34) from the circulation of two normal subjects was rapid with an apparent plasma half-disappearance time (t1/2) between 2 and 3 min; the metabolic clearance rate was 12.9 and 9.0 ml. kg-1 . min-1 respectively. Similarly, the disappearance of endogenous, amino-terminal, immunoreactive PTH from the circulation of two patients with primary hyperparathyroidism after parathyroidectomy was rapid; the apparent t1/2 was approximately 3 min. Homologous amino-terminal specific immunoassays for PTH can thus be useful for the study of both the acute, and chronic, changes of circulating hormone in man and represent an improvement over heterologous unselected assay systems.

Antigenic Activity of Human Urothelial Tissue

Partially purified extracts from 71 human urothelial tumors and from 75 human urothelial non-tumor tissues were used as competing antigens in competition radioimmunoassay in an effort to detect the presence of 1 of thestructural components of type C-ribonucleic acid viruses, the p30 core protein. Testing of the tissue extracts was carried out by homologous and heterologous assay systems using 125I-labeled murine (Friend) and feline (Rickard) p30 antigens and anti-feline and anti-murine p30 antisera. The homologous assays were designed to detect the presenceofp30 antigens in the human tissue extracts having similar characteristics to the p30 antigens of either the feline or murine type C viruses. The heterologous assay system was designed to detect interspecies antigenic determinants common tomurine and feline viruses and also primate viruses. A competing antigen present in 28 per cent of the tissue extracts assayed demonstrated an antigenic protein that competed with the viral p30 protein used in the heterologous radioimmunoassay system and to a lesser extent in the homologous feline radioimmunoassay system. Antigenic competitor proteinswere found in tumor and non-tumor tissue. These data suggest that some human urothelial tissues contain at least part of thegenome of 1 or more type C viruses. Those tissues that are positive only in the heterologous assay system behavelike viral p30 antigens already identified in tissues of several other primates. Those tissues that are positive in the heterologous assay system and the homologous system indicate that the p30 antigenic activity is closely related to that p30 present in feline C-type viruses. Further purification and more detailed characterization of those competing proteins are now under study.

Type-C RNA virus gene expression in human tissue.

Partially purified fractions of human tissues have been analyzed by competition radioimmunoassay for the presence of two of the principle structural components of type-C RNA viruses, the major core protein (p27 to p30) and the major envelope glycopeptides (gp69/71). Screening of tissues was carried out by use of a heterologous assay system of (125)I-labeled Rauscher murine virus p30 antigen and anti-RD 114 virus serum which was found to detect a class of interspecies determinants common to murine, feline, and primate viruses. A competitor with the same apparent affinity for antibody binding as that of purified viral core proteins was found in relatively high concentration in tissues from patients with systemic lupus erythematosus, in some neoplastic tissues, and also in normal human tissues. This competitor from a lupus spleen chromatographed on phosphocellulose and showed size fractionation during gel filtration similar to known p27 to p30 viral proteins. An immunologically reactive protein was also demonstrated by immunodiffusion and by immunoprecipitation of (125)I-labeled human protein with anti-RD 114 p28 serum. Analysis of these human competitor proteins with homologous assay systems of viral core proteins and corresponding antisera showed that all, including the normal tissue extracts, appear similar to core proteins of known viruses, especially the RD 114 and woolly monkey species. A hypothesis suggested by these data is that many, if not all, humans harbor at least part of the genome of one or more type-C viruses, the properties of which are similar to those of viruses from other mammalian species, particularly primates.