Isolation of Com1, a New Gene Required to Complete Meiotic Double-Strand Break-Induced Recombination in Saccharomyces Cerevisiae

We have designed a screen to isolate mutants defective during a specific part of meiotic prophase I of the yeast Saccharomyces cerevisiae. Genes required for the repair of meiotic double-strand breaks or for the separation of recombined chromosomes are targets of this mutant hunt. The specificity is achieved by selecting for mutants that produce viable spores when recombination and reductional segregation are prevented by mutations in SPO11 and SPO13 genes, but fail to yield viable spores during a normal Rec(+) meiosis. We have identified and characterized a mutation com1-1, which blocks processing of meiotic double-strand breaks and which interferes with synaptonemal complex formation, homologous pairing and, as a consequence, spore viability after induction of meiotic recombination. The COM1/SAE2 gene was cloned by complementation, and the deletion mutant has a phenotype similar to com1-1. com1/sae2 mutants closely resemble the phenotype of rad50S, as assayed by phase-contrast microscopy for spore formation, physical and genetic analysis of recombination, fluorescence in situ hybridization to quantify homologous pairing and immunofluorescence and electron microscopy to determine the capability to synapse axial elements.     

Therapy of bovine ocular squamous-cell carcinoma with local doses of interleukin-2: 67% complete regressions after 20 months of follow-up

We have tested the therapeutic potency of peritumorally injected low doses of interleukin-2(IL-2). Seventy tumours of the bovine ocular squamous-cell carcinoma (BOSCC), 1–3 cm in diameter, were treated with 5000, 20 000 or 200 000 U IL-2 from Eurocetus (Chiron) to find the optimal dose for treatment. Injections were given peritumorally on Monday to Friday on 2 consecutive weeks. The size of the tumours was measured before treatment and 1, 3, 4, 9 and 20 months after treatment. After 9 months complete regression was observed in 89% of the tumours treated with 5000 U IL-2, 80% treated with 20 000 U and 67% treated with 200 000 U. After 20 months, there was complete regression of 35%, 31% and 67% of the tumours respectively. The 9-and 20-month results of the 200 000-U treatment are significantly better than those of the 5000-U and 20 000-U treatments taken together. This protocol may be useful to treat advanced inoperable tumours (e.g. of the nasopharynx or skin) of human patients.     

Membrane ultrafiltration: concentration of interleukin-3.

A pilot-plant-scale operation was used for studying membrane ultrafiltration and concentration of kiloliter quantities of the lymphokine interleukin-3 with a single set of membranes. Initial use of ammonium sulfate precipitation of interleukin-3 proved erratic in the recovery of biological activity and resulted in corrosion of the processing equipment. Membrane ultrafiltration proved to be effective in enabling control of the degree of concentration and predicting recovery of the biologically active protein.     

A multigene family of intron lacking and containing genes, encoding for mouse ribosomal protein L7.

Mouse ribosomal protein L7 is encoded by a multigene family. Screening of two mouse genomic libraries with cloned L7 cDNA, has resulted in the isolation of nine independent lambda Charon 4A recombinant phages which include seven different L7 genes. Restriction enzyme mapping of six of these genes (L7-1, L7-16, L7-18, L7-28, L7-35 and L7- 16b ) reveals dissimilarity in sites within the L7 sequences as well as in the flanking regions. Electron microscopic analysis of heteroduplex and S1 nuclease mapping demonstrate that the first five genes contain the entire L7 mRNA sequence but lack introns. Based on these features we propose that these are processed genes. Of the L7 genes described here only one (L7- 16b ) exhibits a high degree of homology with L7 mRNA and contains introns. We discuss the possibility that this low representation of intron containing L7 genes may reflect the proportion of functional L7 genes in this multigene family.     

High-level expression in Escherichia coli of calcium-binding domains of an embryonic sea urchin protein

A plasmid expression vector is described having features that facilitate high-level expression of eukaryotic DNA in Escherichia coli. The vector, designated pMAM17, carries the ColE1 rop gene under the control of the thermally inducible lambda PL promoter. The rop gene product is a negative regulator of ColE1 DNA replication, and its high-level expression is lethal to cells. However, cells harboring a plasmid with an insert in the rop gene grow normally under these conditions. pMAM17 has been used to investigate the properties of a family of proteins expressed in the dorsal ectoderm of sea urchin embryos. The coding sequences of these proteins (termed Spec proteins) have homology to the troponin C superfamily. Large amounts of the Rop-Spec fusion protein were produced at 42 degrees C in E. coli. Unfractionated E. coli extracts containing the fusion protein could be used to produce antibodies that were highly specific for Spec proteins present in crude extracts of sea urchin embryos. Analysis of the Rop-Spec fusion protein on SDS-polyacrylamide gels in the presence and absence of EGTA indicated that the fusion protein bound calcium ions in a manner characteristic of proteins of the troponin C superfamily. This behavior provides biochemical evidence that the Spec proteins are functionally homologous to other members of this superfamily.     

Analysis of intestinal cell proliferation after guanethidine-induced sympathectomy

Summary Guanethidine-induced sympathectomy in the rat during the neonatal period (injection of 20 g/g body weight every 48 h from day of birth until day 14) produces an absolute reduction in the number of sympathetic ganglion cells, but no significant alteration of body weight. Superior cervical ganglia show 79.8 % fewer cell bodies at 15 days and 92.3 % at 45 days; coeliac ganglia exhibit an 81.0 % reduction at 15 days and 89.6 % at 45 days in guanethidine-treated rats as compared to normal controls. The sympathetic ganglion cells that remain after treatment have an abnormal morphological appearance with distended mitochondria and depletion of endoplasmic reticulum. Sympathectomy produces a prolongation of the generation cycle time (Tc) as measured by the colchicine-induced mitotic arrest technique, and a decrease in labelling, mitotic, and migration indices. In addition, sympathectomy suppresses the amplitude of the circadian rhythm in mitotic activity. The general suppression of this activity in the intestinal epithelium is more pronounced in the jejunum and ileum than in the duodenum. Variation in the effectiveness of sympathectomy on the inhibition of intestinal cell proliferation may be related to segmental differences in cell proliferation, to segmental differences in innervation, and/or to segmental variation in the effectiveness of guanethidine.Supported by N.I.H. grant DE04557 to R.M.K. and N.I.H. grant 5-SO1-RR5373 to the University of Kansas Medical Center. The authors wish to acknowledge Charles A. Brownley, CIBA-Geigy, Summit New Jersey, U.S.A. for the gift of guanethidine-sulfate     

Therapeutic application of various immunostimulators on the L2C guinea-pig leukemia

Summary Immunostimulators such as Corynebacterium parvum (C. parvum), Bacillus Calmette-Guerin (BCG), pyran copolymer, and glucan were examined in the guinea pig L ₂ C lymphoblastic leukemia model to determine their capacity for therapeutic modulation of the immune response of the host toward controlling leukemic cell proliferation. The dose, route, and frequency of administration of the stimulators were also evaluated as a function of time in order to obtain an optimal antileukemic effect. Results indicated that only C. parvum and BCG were capable of significantly increasing host survival when given 1 day after an inoculation of 1.5×10 ⁴ viable leukemic cells. Administration of BCG or C. parvum, alone or in combination with irradiated blast cells on either days 4 or 7, was totally ineffective in prolonging survival. In the majority of cases, enhanced leukemic growth was observed on these days. The combination of BCG and/or C. parvum with irradiated syngeneic blast cells given 24 h after leukemia inoculation promoted a synergistic response with a significant increase in median survival time and a number of long-term survivors.This work was supported by contract N01-CP-53566 within the Virus Cancer Program of the National Cancer Institute     

The influence of some prostaglandins on DNA synthesis and DNA excision repair in mouse spleen cells in vitro

$\text{In vitro}$ experiments were performed on mouse spleen cells to establish possible influences of some naturally occurring prostaglandins on DNA synthesis and DNA excision repair. The prostaglandins A₁, B₁, E₁, E₂ and F₂ were tested in concentrations of lopg, 5ng and 2.5μg per ml cell suspension. DNA synthesis was significantly increased by pgF_2α in all the three concentrations tested, while the other tested prostaglandins were essentially ineffective. DNA excision repair was significantly inhibited by PgF₁ and PgF₂ at 5ng/'ml and at 2.5μg/ml but increased by PgF_2α in the two lower concentrations. The rejoining of DNA-strand breaks after γ-irradiation was slightly reduced by PgF₁, PgE₂ and PgF_2α at 2.5μg/ml.