Use of peptide tagging to detect proteins expressed from cloned genes: deletion mapping functional domains of Drosophila hsp 70.

We have developed a technique which allows specific detection of proteins expressed from cloned genes. The method involves fusion of an oligonucleotide coding for part of the neuropeptide substance P to the 3' end of the gene; the protein can then be detected with a monoclonal antibody that recognises this peptide. We have used this method to determine the properties of deletion mutants of the major Drosophila heat shock protein, hsp70, expressed in monkey COS cells. The results suggest that this protein has two distinct domains. Both are capable of accumulating in the nucleus of unstressed cells, but only the more highly conserved N-terminal domain is able to bind to nucleoli following a heat shock. This implies that nucleolar binding and nuclear migration are distinct properties of the protein, and suggests that the former may be of functional importance. In addition, we observed a novel effect of heat shock on cellular metabolism: protein fragments that are normally rapidly degraded are stabilized. The effect persists for several hours after the heat shock, but does not require expression of heat shock proteins. Together with previously published data, these results suggest an intimate relationship between protein degradation and the heat shock response.     

Conservation in rat liver of light and heavy subunit sequences of mammalian ferritin. Presence of unique octopeptide in the light subunit

Ferritin, an iron-storage protein found in all life forms examined, is composed of varying proportions of two subunits of different molecular weight, heavy (H) and light (L). Using cDNA clones, we have determined the nucleotide sequence corresponding to the mRNA of the L-subunit of rat liver ferritin. The coding region of 546 nucleotides (182 amino acids) is flanked by 5'- and 3' -untranslated regions of approximately 130 and 150 nucleotides, respectively. The rat liver L-subunit amino acid sequence derived from the reading frame of the cDNA showed 88% and 82% homology, respectively, with the amino acid sequences of horse spleen ferritin (Heusterspreute, M., and Crichton, R. R. (1981) FEBS Lett. 129, 322-327), and human spleen ferritin (Wustefeld, C., and Crichton, R. R. (1982) FEBS Lett. 150, 43-48), thus demonstrating evolutionary conservation of the L-subunit sequence. However, a major difference between the rat and the horse and human sequences is the insertion of an octopeptide near the COOH-terminus of the rat protein resulting in a slightly longer peptide chain in this species. The reading frame and parts of the derived amino acid sequence including the octopeptide sequence were confirmed by direct amino acid sequencing of cyanogen bromide peptides from rat liver ferritin. Minor fragments of rat liver ferritin, presumably derived from the H-subunit, were also isolated after cyanogen bromide treatment. On sequencing, these H-peptides showed limited homology with regions of the L-sequence but extensive homology with published H-sequences from human liver and spleen. The H-subunit sequence did not contain the octopeptide found as part of the L-subunit sequence.     

Ovarian and endocrine responses in the cat after coitus

LH release leading to ovulation was induced in 17 of 29 oestrous periods. The time of ovulation after coitus was determined by histological examination or by observation at laparotomy of ovaries in situ. Histological methods revealed that ovulation was complete in most follicles (9 of 13) at 32 h post coitum and in all follicles that were involved in the ovulatory process by 36 h. When laparotomy was used, no signs of preovulatory change were noted at the first observation time, 22 h post coitum, but in 4 cycles in which the entire process of ovulation was observed, the ovulatory process occurred between 23 and 28 h (3 follicles), 23 and 27 h (2 follicles), 25 and 28 h (3 follicles), and 25 and 29 h (3 follicles) post coitum. The first ovulatory process noted was complete at 25 h post coitum. In cats, LH release continued over a 16-h period before returning to baseline (long surge), values being 616 +/- 180 ng/ml at 1/2 h and 941 +/- 154 ng/ml at 2 h post coitum. In 6 cats the LH release pattern was limited to a 4-h period (short surge), values being 537 +/- 218 ng/ml at 1/2 h and 353 +/- 245 ng/ml plasma at 2 h and basal (49 +/- 18 ng/ml) by 4 h post coitum. Decreased secretion of oestrogen by follicles in animals undergoing ovulation was first observed at 16 h post coitum. It is concluded that coitus induces LH release within minutes in the cat and that ovulation begins about 24 h later and finishes by about 32 h post coitum. Only one coital input can cause LH release for as long as 16-20 h although shorter periods of LH release (4 h or less) can result in ovulation.     

A model for generalization and specification by single neurons

A rule for environmentally dependent modification of the neuronal state is examined. Under the rule, the neuron selects a trigger feature that matches either a particular pattern in the stimulus set, or the most common pattern component, depending on a certain parameter. Thus a neuron may evolve to respond to its stimulus environment in one of two capacities, namely specification or generalization. Neurons of the former variety are labelled S-cells; and those of the latter, G-cells. In the model, synaptic modification is modulated by two postsynaptic mechanisms which act antagonistically to strengthen or weaken the synaptic connectivities. The functional dependence of these mechanisms on the postsynaptic activity is shown to determine whether the neuron acts as an S-cell or a G-cell. A circuit is proposed for a module that consists of a G-cell and several S-cells sharing a common set of inputs. By inhibiting the G-cells, the S-cell acts as a contrast-enhancing element, increasing their specificities for individual patterns in the stimulus set. The output from the module is a recoded representation of the environment with respect to its general and distinctive features.This work was supported in part by United States Office of Naval Research Contract N00014-81-K-0136     

Centromeres Reposition to the Nuclear Periphery during L6E9 Myogenesisin Vitro

To test the hypothesis that genome architecture in interphase is related to nuclear function, we have compared the disposition of centromeres in nuclei of undifferentiated rat L6E9 myoblasts with that in nuclei of L6E9 myotubes differentiatedin vitro.Immunofluorescence labeling showed that centromeres repositioned to the nuclear periphery during differentiation, and condensed chromatin was more prominent at the myotube nuclear envelope by electron microscopy. These data indicate that, in parallel with considerable changes in gene expression, the spatial order of the genome undergoes substantial rearrangement during myogenesis.