Two Novel Endopeptidases Released into the Medium during Mating of Gametes of Chlamydomonas reinhardtii

During the mating reaction between mt⁺ and mt^- gametes of Chlamydomonasreinhardtii, two novel endopeptidases, each of which was ableto digest the B chain of insulin, were released into the culturemedium, together with a gamete lytic enzyme (GLE) which is responsiblefor digestion of the gametic cell walls. Both endopeptidasesand GLE were copurified from the mating medium by column chromatographyon DEAE-cellulose and concanavalin A. Gel filtration separatedthe peptidases, which were unable to digest gametic cell walls,into two fractions, endopeptidase-1 and endopeptidase-2. Theseenzymes were also separated from GLE, which was unable to digestthe B chain of insulin. Endopeptidase-1, with a molecular massof about 200 kDa, cleaved the B chain of insulin at the Ala¹⁴-Leu¹⁵peptide bond, and this activity was inhibited by EDTA. Endopeptidase-2,with a molecular mass of about 110 kDa, digested the peptideat the Leu¹⁵-Tyr¹⁶ peptide bond and was sensitive to iodoacetateand chymostatin. When the cell walls of gametes of either mating-typewere digested prior to mating with exogenously added GLE, thetwo endopeptidases were released into the medium, a result thatsuggests that they are stored, like GLE, outside the plasmalemma. (Received March 25, 1994; Accepted June 13, 1994)     

A Genetic Linkage Map of the Mouse Using Restriction Landmark Genomic Scanning (Rlgs)

We have developed a multiplex method of genome analysis, restriction landmark genomic scanning (RLGS) that has been used to construct genetic maps in mice. Restriction landmarks are end-labeled restriction fragments of genomic DNA that are separated by using high resolution, two-dimensional gel electrophoresis identifying as many as two thousand landmark loci in a single gel. Variation for several hundred of these loci has been identified between laboratory strains and between these strains and Mus spretus. The segregation of more than 1100 RLGS loci has been analyxed in recombinant inbred (RI) strains and in two separate interspecific genetic crosses. Genetic maps have been derived that link 1045 RLGS loci to reference loci on all of the autosomes and the X chromosome of the mouse genome. The RLGS method can be applied to genome analysis in many different organisms to identify genomic loci because it used end-labeling of restriction landmarks rather than probe hybridization. Different combinations of restriction enzymes yield different sets of RLGS loci providing expanded power for genetic mapping.     

Ubiquitin and ubiquitin-protein conjugates in PC12h cells: Changes during neuronal differentiation

Ubiquitin and ubiquitin-protein conjugates in PC12h cells were detected with in vitro [¹²⁵I]ubiquitination, and quantified by immunoblotting. These levels were altered by nerve growth factor (NGF), which promotes neuronal differentiation. (i) Levels of high molecular weight (HMW) ubiquitin-protein conjugates ranging from 40 to 1,000kDa were increased by 2 days of NGF treatment, and remained high up to 10 days of NGF treatment. (ii) Ubiquitin and a 23-kDa conjugate tended to be decreased from days 2 to 10 of NGF treatment. 10-Day culture with 10 nM staurosporine, an protein kinase inhibitor, that blocks NGF-induced neurite outgrowth suppressed the NGF-induced increases in levels of HMW conjugates. Cyclic AMP and forskolin, both of which promote neurite outgrowth, mimicked the NGF-induced changes in ubiquitin and HMW conjugates, but phorbol ester and epidermal growth factor had little effect. These findings suggest that changes in ubiquitin-protein conjugates are closely coupled with neuronal differentiation.     

A meiotic DNA polymerase from Coprinus cinereus: further purification and characterization

A meiotic DNA polymerase that is present at a high level of activity in meiotic cells of a basidiomycete, Coprinus cinereus, was purified to near homogeneity using synthetic RNA homopolymer [poly(C)] cellulose column chromatography. This report presents the first extensive purification and characterization of any eukaryotic DNA polymerase having a role in meiosis. This enzyme is a single polypeptide with a molecular mass of 65,000. Activity in this enzyme requires magnesium ions and occurs at an optimal pH of 7.5. It is strongly inhibited by dideoxythymidine triphosphate but is relatively insensitive to aphidicolin and N-ethylmaleimide and can use poly(C)/oligo(dG)_12–18 as a template-primer. Polymerase activity can be found only in cells at meiotic prophase, even though the enzyme has been identified in somatic cells in an inactive state using immunoblot analysis. Its distinctive distribution makes possible a genetic and biochemical analysis of functional role of a meiotic DNA polymerase in meiotic recombination, repair and synthesis.Abbreviations ddTTP 2,3-dideoxythymidine 5-triphosphate - NEM N-ethylmaleimide - PMSF phenylmethylsulfnylfluoride - BSA bovine serum albumin     

A tumor cell line producing granulocyte colony-stimulating factor and an immune suppressive factor

From a patient, both a cell line incapable of secreting granulocyte colony-stimulating factor (G-CSF) (TC873) and a cell line capable of secreting G-CSF (TCM902) were established. The effector cells induced, with TC873 cells showed a high lytic capacity against two types of tumor cells. The effector cells induced by TCM902 cells did not show such capacity. Furthermore, the TCM902 cells excreted a factor suppressing the proliferation of lymphokine activated killer (LAK) cells and the autologous tumor cell lysis of tumor associated lymphocytes. This factor probably is TFG- ₁.Abbreviations CSF colony stimulating factor - ELISA enzyme-linked immunosorbent assay - G granulocyte - GM granulocyte-monocyte - IFN interferon - IL interleukin - LAK lymphokine activated killer - M monocyte - MLTC mixed lymphocyte tumor cell culture - TGF transforming growth factor - TILs tumor infiltrating lymphocytes - TNF tumor necrosis factor     

New format for the chromosome committee reports

Editorial Note

New format for the chromosome committee reports     

Effects of adaptive predatory and anti-predator behaviour in a two-prey—one-predator system

Summary Two prey populations that share a common predator can interact indirectly by causing changes in the predator's foraging behaviour. Previous work suggests that adaptive choice of prey by the predator usually has two related consequences: (i) the predation rate on a particular prey species increases with the relative and/or absolute abundance of that prey; and (ii) increases in either prey population produce a short-term increase in the fitness of the other prey (short-term indirect mutualism between prey). This paper investigates how these two consequences are changed if the prey exhibit adaptive anti-predator behaviour. In this case, the predation rate on a particular prey often decreases as the prey's density increases. The predator then usually exhibits negative switching between prey. However, the presence of adaptive antipredator behaviour does not change the short-term mutualism between prey. In this case, as a prey becomes less common, it achieves a larger growth rate by reducing its anti-predator effort. These results imply that observations of the relationship between prey density and predation rate cannot be used to infer the nature of the behavioural indirect effect between prey that share a predator.     

Both the SH2 and SH3 domains of human CRK protein are required for neuronal differentiation of PC12 cells.

Human CRK protein is a homolog of the chicken v-crk oncogene product and consists mostly of src homology region 2 (SH2) and SH3, which are shared by many proteins, in particular those involved in signal transduction. SH2 has been shown to bind specifically to phosphotyrosine-containing peptides. We report here that both SH2 and SH3 are required for signaling from CRK protein. Microinjection of the CRK protein induced neurite formation of rat pheochromocytoma cell line PC12. This activity was abolished by mutation of the CRK protein in either SH2 or SH3. The neuronal differentiation induced by the CRK protein was blocked by an excess amount of peptides containing CRK SH3. Moreover, we identified three proteins, of 118, 125, and 136 kDa, which bound specifically to CRK SH3. The CRK-induced neuronal differentiation was also suppressed by monoclonal antibodies against either CRK SH2 or p21ras. These results suggest that both SH2 and SH3 of the CRK protein mediate specific protein-protein binding and that the resulting multimolecular complex generates a signal for neurite differentiation through activation of p21ras.