Multiple octamer binding sites in the promoter region of the bovine alpha s2-casein gene.

Using a set of overlapping oligonucleotides from the promoter region of the bovine alpha s2-casein gene we have identified two nuclear factors which probably are involved in expression of this gene and the related calcium sensitive alpha s1- and beta-casein genes. One of these factors which was present in extracts of all tissues that have been tested including Hela cells turned out to be the octamer binding protein OCT-1. Oct-1 binds with different affinity to 4 sites at positions centred around -480, -260, -210 and -50. The strongest of these 4 binding sites, the one around position -50, is highly conserved in all calcium sensitive caseins of mouse, rat, rabbit and cattle. The other nuclear factor (MGF, mammary gland factor) which is specifically expressed in the mammary gland, binds to a site around position -90. This binding site is also highly conserved in all calcium sensitive caseins of mouse, rat, rabbit and cattle.     

Rat prostatic growth factors: purification and characterization of high and low molecular weight epidermal growth factors from rat dorsolateral prostate

Growth factors which possibly participate in androgen-induced proliferation of rat prostate epithelial cells have been purified and characterized. Four distinct forms of growth factor were found in the extract of rat dorsolateral prostate. One of the factors was a member of heparin-binding growth factor (HBGF) family judging from its high affinity for heparin-Sepharose. The other three factors were capable of competing with [125I]epidermal growth factor (EGF) for the cell surface receptor, and recognized by anti-rat EGF antiserum. These EGF-like factors (EGF1-EGF3) were purified by ion-exchange chromatography, gel filtration and reverse phase HPLC. EGF1 showed microheterogeneity on chromatographic and electrophoretic separation and N-terminal sequence analysis. EGF1 showed an average molecular weight of about 35,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. These results indicated that EGF1 was a mixture of high molecular weight forms of EGF. The molecular weights of EGF2 and EGF3 were similar to that of rat submaxillary gland EGF (Mr = 5400). The amino acid sequence of EGF2 was identical with that of rat EGF except for the N- and C-terminal amino acids: aspartic acid instead of asparagine was found at the N-terminal position and C-terminal arginine was missing in EGF2. Although the N-terminal sequence of EGF3 (1-19) was identical with that of EGF2, the two factors were completely separated by gel filtration indicating a difference in the C-terminal structure. EGF1, EGF2 and EGF3 but not HBGF stimulated proliferation of primary cultured rat dorsolateral prostate epithelial cells.     

Strain Difference in the Up-Regulation of FGF-2 Protein Following a Neurotoxic Lesion of the Nigrostriatal Pathway

Lesions of the nigrostriatal pathway are known to induce a compensatory up-regulation of various neurotrophic factors. In this study we examined protein content of basic fibroblast growth factor (FGF-2) in tissue samples taken from the ventral midbrain and striatum at two different time points following a neurotoxic lesion of the nigrostriatal pathway in two different rat strains, the outbred Sprague–Dawley (SD) and inbred F344 × Brown Norway F1 hybrid (F344BNF₁). Despite both rat strains having comparable lesions of the nigrostriatal pathway, we observed a difference in the temporal up-regulation of FGF-2 in ventral midbrain samples taken from the side ipsilateral to the lesion. Basic FGF was significantly up-regulated in ventral midbrain in SD rats 1 week post-lesion while we did not observe an up-regulation of FGF-2 in the lesioned ventral midbrain of F344BNF₁ at this same time point. However, both strains showed a significant up-regulation of FGF-2 in the lesioned ventral midbrain 3 weeks post-lesion. Sprague–Dawley rats also appeared to be more sensitive to the lesion in terms of up-regulating FGF-2 expression. The differences reported here suggest currently unknown genetic differences between these two strains may be important factors for regulating the compensatory release of neurotrophic factors, such as FGF-2, in response to a neurotoxic lesion of the nigrostriatal pathway.     

Effects of neonatal undernutrition on the electrocortical development of the association areas in the rat

The electrocortical effects provoked by neonatal undernutrition and the environmental sensorial stimuli were studied in the cortical association areas of developing Wistar rats. When the interaction between these two factors was interfered (Experiment 1), the average frequency of the ECoG in the early starved rats was significantly increased than controls. Moreover, if these two factors were combined (Experiment 2) not significant differences in the ECoG average frequencies were observed. The data suggest that the maturation of cells underlying the ECoG in the association areas of the rat, requires not only an adequate supply of nutrients, but also the influence of sensory cues arising from the mother, littermates and the environmental surrounding.     

Ribosome specificity of archaebacterial elongation factor 2. Studies with hybrid polyphenylalanine synthesis systems

Polyphenylalanine synthesis with ribosomes and two separated, partially purified elongation factors (EF) was measured in cell-free systems from the archaebacteria Thermoplasma acidophilum and Methanococcus vannielii, in an eukaryotic system from rat liver and an eubacterial one with Escherichia coli ribosomes and factors from Thermus thermophilus. By substitution of heterologous EF-2 or EF-G, respectively, for the homologous factors, ribosome specificity was shown to be restricted to factors from the same kingdom. In contrast, EF-1 from T. thermophilus significantly cooperated with ribosomes from T. acidophilum.     

Systematic binding analysis of the insulin gene transcription control region: insulin and immunoglobulin enhancers utilize similar transactivators.

The 5' regulatory region (-345 to +1) of the rat insulin I gene (Ins-I) was examined for binding to cellular factors with short oligodeoxynucleotide probes. Over 40 binding species were detected. The binding profiles were specific for each cell type studied. We characterized the factors binding two elements crucial for enhancer activity (the Nir and Far boxes) which bear sequence similarity to the microE1, microE2, and microE3 elements of the immunoglobulin heavy-chain enhancer. The Nir box binds three cellular factors that display preferential affinities for microE1, microE2, or microE3, and the Far box binds two factors related to microE2 or microE3. The insulin gene enhancer was mutated at the Nir box element to reflect the sequences of microE1, microE2, or microE3. Ins-microE2 was fully active, Ins-microE3 was partially active, and Ins-microE1 was inactive. Thus, factors similar or identical to nuclear factor NF-microE1, NF-microE2, or NF-microE3 may play a role in the activity of the insulin gene enhancer.     

Partial purification and properties of the cytoplasmic factors modulating adenylate cyclase activity in rat lung plasma membranes

The adult rat lung supernatant contains some factors which markedly enhance adenylate cyclase activity in membranes (Nijjar, M.S. (1979) Biochim. Biophys. Acta 584, 43–50). These factors were separated into two less active components (peaks 1 and 2) by DEAE-cellulose chromatography. However, their recombination restored the full activation of adenylate cyclase. Further purification and characterization of these factors revealed that the activation in peak 1 contained two proteins of low (14 500) and high (65 000) molecular weight whereas the activator in peak 2 contained only one protein of 65 000. The kinetics of adenylate cyclase activation revealed that both the K_m and V values were affected. The data also demonstrate that calmodulin was not involved in the cytoplasmic activation of adenylate cyclase in rat lungs.     

Evolution of the rat immunoglobulin gamma heavy-chain gene family

The sequences of the four immunoglobulin gamma heavy chains of the rat (gamma 1, gamma 2a, gamma 2b, gamma 2c) have been determined. These sequences reveal that the rat genes have evolved differently from the closely related mouse gamma genes (gamma 1, gamma 2a, gamma 2b, gamma 3): in rat two of the four genes (gamma 2a and gamma 1) are 94% homologous to each other and best resemble the single mouse gamma 1 gene. Rat gamma 2b is equivalent to the mouse gamma 2a/gamma 2b pair as regards both nucleotide sequence and antibody effector functions whilst rat gamma 2c resembles mouse gamma 3. In evolutionary terms this suggests the existence of a set of three common C gamma genes before separation of rat and mouse as individual species. In addition, two independent duplication events must have occurred after species separation affecting different constant regions; this yielded rat gamma 2a and gamma 1 as a recently evolved pair and mouse gamma 2a and gamma 2b as a different pair. Furthermore, the sequence comparisons reveal several other features of interest; rat IgG2b lacks two amino acids in CH1 which are conserved in all other sequenced gamma chains. Residues believed to be essential for monocyte interaction (FcRI) are retained only in rat gamma 2b and not in the other rat gamma genes whilst a particular motif involved in C1q interaction shows a variation in both rat IgG1 and rat IgG2a which has not been observed previously.     

Monoclonal antibody detection of transformation associated protein (TAP) in ts110 Moloney murine sarcoma virus transformed 6M2 cells that are different from the mos gene product

By the use of a highly specific monoclonal antibody (designated MC), we were able to detect three radiolabeled bands with molecular weights of 60,000, 63,000, and 66,000 daltons in the ts-110 Moloney murine sarcoma virus mutant-transformed rat kidney cells known as 6M2. Expression of transformation properties as well as these three bands in 6M2 cells was found to be temperature sensitive. Therefore, MC detected factors that are apparently associated with the transformation of 6M2 cells. These factors are tentatively referred to as transformation associated proteins. These transformation proteins were found in two other Moloney murine sarcoma virus-transformed rat cell lines. These proteins were found to differ from known gene products of the ts-110 Moloney murine sarcoma virus mutant and do not have kinase activity. The transformation associated proteins may represent rat cellular factors activated during the transformation of rat cells by Moloney murine sarcoma virus.     

Regulation of the rat CYP4A2 gene promoter by c-Jun and octamer binding protein-1

The physiologically important cytochrome P450 (CYP) 4A2 arachidonic acid omega-hydroxylase gene is widely expressed in rat tissues. Although the induction of CYPs 4A by peroxisome proliferators and dietary lipids is established there is minimal information on the factors that control constitutive expression. To address this issue we cloned 1.4 kb of the CYP4A2 5'-upstream region and identified several DNA elements that resembled the activator protein-1 (AP-1) consensus sequence. Using a series of 5'-truncated reporter constructs a 42 bp region was detected that was responsive to the AP-1 factor c-Jun, which is important in basal gene regulation. The roles of two putative AP-1 elements at -47/-41 and -31/-25 were tested, with the former emerging from studies with mutagenised constructs as the functionally important site. These findings were supported by electromobility shift assay (EMSA) studies that indicated the interaction of the -47/-41 element with c-Jun. The -31/-25 element mediated the suppression of CYP4A2 transactivation by octamer binding protein-1 (oct-1). Thus, mutagenesis of this element relieved the modulatory effect of oct-1 on c-Jun-mediated transactivation. In EMSAs, the binding of nuclear proteins to the -31/-25 element was competed by an oct-1 consensus sequence and supershifted by an anti-oct-1 antibody. Overexpression of c-Jun in rat liver-derived H4IIE cells increased CYP4A2 mRNA to approximately 2-fold of control, but oct-1 overexpression was without significant effect. From chromatin immunoprecipitation assays both c-Jun and oct-1 bound to the CYP4A2 5'-upstream sequence in H4IIE cells. These findings implicate c-Jun and oct-1 as potentially important constitutive factors that modulate the transactivation of the CYP4A2 gene promoter.