Structure of yeast pGKL 128-kDa killer-toxin secretion signal sequence. Processing of the 128-kDa killer-toxin-secretion-signal-alpha-amylase fusion protein.

The linear double-stranded DNA plasmid pGKL1 in yeast encodes a killer toxin consisting of 97-kDa, 31-kDa and 28-kDa subunits. A 128-kDa protein precursor of the 97-kDa and 31-kDa subunits, was first synthesized with a 29-amino-acid extension at its NH2-terminus as a secretion signal sequence. In the present study, the property of this signal sequence was studied by the analysis of a fusion protein with mouse alpha-amylase. Using the secretion signal sequence of the killer protein, the mouse alpha-amylase was successfully secreted into the culture medium. An intracellular precursor form of alpha-amylase was identified and purified. Analysis of the NH2-terminal sequence of this precursor molecule indicated that it corresponded to the secretory intermediate (pro form) of alpha-amylase with the removal of the hydrophobic segment (Met1-Gly16) of the secretion signal. Both the secretion of alpha-amylase into the culture medium and the detection of the pro-alpha-amylase species in the cells were prohibited by a sec 11 mutation, or by the conversion of Gly to Val at the 16th position of the secretion signal. These results strongly suggest that the cleavage occurs between Gly16 and Leu17 by a signal peptidase, and that this cleavage is required for the secretion of alpha-amylase into the medium. Based on the data from the NH2-terminal amino acid sequences of secreted alpha-amylases, we conclude that the 29-amino-acid secretion signal present in the 128-kDa killer toxin precursor protein is a prepro structure.     

Induction of cytochrome P-448 isozyme(s) in primary cultured rat hepatocytes by drugs which induce different isozymes in vivo

Effect of 2-methoxy-4-aminoazobenzene (2-MeO-AAB) and 3-methylcholanthrene (MC) on the induction of microsomal cytochrome P-448 isozymes in primary cultured rat hepatocytes was examined by means of immunochemical methods such as protein A-enzyme-linked immonosorbent assay and immuno-blots using anti-rat cytochrome P-448 monoclonal antibodies and by means of bacterial mutation tests. Although 2-MeO-AAB selectively induced cytochrome P-448H and MC induced both cytochrome P-448H and a low spin form of cytochrome P-448 (P-448L) in the liver of rats, addition of these chemicals to primary cultured rat hepatocytes resulted in selective induction of cytochrome P-448L, as determined by the immunological methods. This was substantiated by the bacterial mutation test using Salmonella typhimurium TA 98 bacteria and two aromatic amine substrates with different specificities to the cytochrome P-448 isozymes. These results suggest that the responses of rat hepatocytes to cytochrome P-450 inducers are different in in vivo and in vitro.     

Isolation and Characterization of Mutants Which Show an Oversecretion Phenotype in Saccharomyces Cerevisiae

We have isolated mutants responsible for an oversecretion phenotype in Saccharomyces cerevisiae, using a promoter of SUC2 and the gene coding for alpha-amylase from mouse as a marker of secretion. These mutations defined two complementation groups, designated as ose1 (over secretion) and rgr1 (resistant to glucose repression). The ose1 mutant produced an oversecretion of amylase by 12- to 15-fold under derepressing conditions; however, the amylase mRNA was present at nearly the same amount as it was in the parent cells. No expression of the amylase gene was detected under repressing conditions. The rgr1 mutant oversecreted amylase by 11- to 13-fold under repressing conditions by 15- to 18-fold under derepressing conditions. The rgr1 mutant showed pleiotropic effects on the following cellular functions: (1) resistance to glucose repression, (2) temperature-sensitive lethality, (3) sporulation deficieny in homozygous diploid cells, and (4) abnormal cell morphology. The rgr1 mutation was not allelic with ssn6 and cyc9, and failed to suppress snf1.     

The yeast linear DNA killer plasmids, pGKL1 and pGKL2, possess terminally attached proteins.

The terminal structures of linear DNA killer plasmids from yeast, pGKL1 and pGKL2, were analyzed. Results obtained by exonuclease treatments of these plasmids show that both pGKL plasmids have free hydroxyl 3'-ends and blocked 5'-ends. Electrophoretic analysis of the terminal restriction fragments treated with proteases revealed that pGKL1 and pGKL2 have proteins bound at 5'termini and that the terminal protein of pGKL1 is distinct from that of pGKL2. This is the first linear DNA-terminal protein association found in yeast.     

Effects of various electric fields on the fusion and in vitro development of mouse two-cell embryos

This study was undertaken to examine the effects of various electric fields such as alternating current (a.c.) voltage, fusion pulse strength, pulse duration, pulse number and electrode geometry on blastomere fusion and developmental rates of mouse two-cell embryos. The a.c. voltages (6 and 12 V/mm) did not affect the fusion and developmental rates. High fusion and developmental rates were obtained when pulse strengths of 1.0 to 2.5 kV/cm, pulse durations of 30 to 90 mu sec and pulse numbers of 1 to 6 were applied using a wire chamber. Comparison of electrode geometries showed that fusion rates were similarly high (93 to 98%) when pulse strengths of 1.0 to 2.5 kV/cm were applied, regardless of the electrode geometry. However, significantly lower developmental rates were observed in a rectangular chamber compared with those in a wire chamber, except when the pulse strength was 1.0 kV/cm. It was further observed that in a rectangular chamber, the developmental rate decreased with increasing pulse strength from 1.0 to 2.0 and 2.5 kV/cm. The results of this study indicate that by using a wire chamber, electric fields can be successfully applied across a relatively wide range of pulse strength, duration and number to provide sufficiently high fusion and subsequent developmental rates. The fusion conditions did, however, vary with chambers of different electrode geometries.     

Cloning of a glycine receptor subtype expressed in rat brain and spinal cord during a specific period of neuronal development.

Complementary (c) DNAs encoding a glycine receptor (GlyR) isomer were cloned from libraries constructed in lambda ZAPII with poly (A)+ RNA of neonatal rat spinal cord. Northern blot analysis revealed that RNA hybridized to the cloned cDNA is detectable only for a period of late embryonic/early postnatal stage of the spinal cord. Moreover, other central nervous tissues, such as hippocampus and cerebral cortex, in the infant rats are also rich in this message. The 'neonatal (N) GlyR' has 71% homology to that of another GlyR isoform in which adult rad cord is rich (AGlyR). Injection of a single RNA transcribed from the NGlyr-cDNA into Xenopus oocyte induced functional formation of glycine-gated Cl- channels, however, its pharmacological property differed from that of AGlyR.     

Modulation of the immune response to tumors by a novel synthetic compound, (4R)-3-benzoyl-N-[(1R)-1-phenylethyl]-4-thiazolidinecarboxamide (RS-0481)

Summary RS-0481, (4R)-3-benzoyl-N-[(1R)-phenylethyl]-4-thiazolidinecarboxamide, is a compound that can re-establish the function of certain lymphoid cell populations impaired by the presence of a growing tumor in an animal. The compound markedly augmented the tumorspecific cytotoxic T lymphocytes,Tdth (delayed-type hypersensitivity T cells), and the nonspecific lymphokine-activated-killer-cell-like cell responses. It also enhanced the tumor-inhibitory effect of macrophages in tumor-bearing mice, but not in normal mice, indicating that it enhances the antitumor immune responses. Lymphocytes from RS-0481-treated tumor-bearing mice released significantly higher amounts of macrophage-activating factor(s) (MAF) and interleukin-2(IL-2)-like factors in culture compared with lymphocytes from untreated animals. Also, sera from treated tumor bearers showed elevated colony-stimulating factor (CSF) activity. Although the compound did not influence the factor-producing activity in mice without tumor, it enhanced the responsiveness of their bone marrow cells, T cells, and macrophages to CSF, IL-2, and MAF. It seems therefore possible that the compound enhances the responsiveness of immunocompetent cells to cytokines, resulting in a marked augmentation of antitumor T cell responses in tumor-bearing mice. Consistently it inhibited the development of lymph node metastasis of transplanted X5563 plasmacytoma, and we showed that T cells play a decisive role in this inhibition. The compound also counteracted the development of suppressor T cell activity in the spleen of tumor-bearing mice.     

Separation and concentration of Δ-6-keto-PGF1α using monoclonal antibody to ω3-olefin structure of trienoic prostanoids

A monoclonal antibody against cis-3-hexen-1-ol was prepared and used to separate and/or concentrate Δ¹⁷-6-keto-prostaglandin F_1α (PGF_1α) in the human sera. cis-3-Hexen-1-ol was conjugated with the human serum albumin (HSA) according to the N-succinimidylester method and hyperimmunized to BALB/c mouse. The monoclonal afntibodies were obtained from hybridoma clones established by a fusion between SP2/0-Ag14-k13 mouse myeloma cells and splenocytes of a mouse. A monoclonal antibody, named 4G9-12B, recognized the epitope characteristic for ω3-olefin structure. The 4G9-12B antibody became more specific for Δ¹⁷-6-keto-PGF_1α than 6-keto-PGF_1α by applying inhibition ELISA using amino-residue coating plates. Using the prepared immunoaffinity columns of this antibody, Δ¹⁷-6-keto-PGF_1α was clearly detected in 6 pg/ml of the human blood sera by GC/MS analysis. These results suggest that the monoclonal antibody to the partial structure of trienoic prostanoid, ω3-olefin unit, and that its immunoaffinity columns are useful in separating and concentrating Δ¹⁷-6-keto-PGF_1α in the human blood or urine.     

Integration of heterologous genes into the chromosome of Saccharomyces cerevisiae using a delta sequence of yeast retrotransposon Ty

Summary Distribution of a delta () sequence of the Ty element on a chromosome of the yeast Saccharomyces cerevisiae was analysed by pulsed-field gel electrophoresis. More than 100 copies of the sequence were non-randomly distributed on the chromosome. Using the sequence as a recombination site, mouse -amylase and human -endorphin genes were introduced into the chromosomal DNA. The integration occurred on a particular chromosome in each case and the copy number was estimated as three to five. It was also found that single- or multi-copy integration occured at a single or multiple sites on the particular chromosome. The integrants secreted -amylase and -endorphin by three-to fivefold compared with single-copy integrants. This type of integration was mitotically stable over a period of 50 generations under non-selective conditions. Offprint requests to: A. Sakai     

Nucleotide sequence analysis of avian retroviruses: structural similarities with transposable elements

Integrated retroviral genomes are flanked by direct repeats of sequences derived from the termini of the viral RNA genome. These sequences are designated long terminal repeats (LTRs). We have determined and analyzed the nucleotide sequence of the LTRs from several exogenous and endogenous avian retroviruses. These LTRs possess several structural similarities with eukaryotic and prokaryotic transposable elements: 1) inverted complementary repeats at the termini, 2) deletions of sequences adjacent to the LTR, 3) small duplications of host sequences flanking the integrated provirus, and 4) sequence homologies with transposable and other genetic elements. These observations suggest that LTRs function in the integration and perhaps transposition of retrovirus genomes. Evidence exists for the presence of a strong promoter sequence within the LTR. The retroviral LTR also contains a "Hogness box" up-stream of the capping site and a poly(A) signal. These features suggest an additional role for the LTR in the regulation of gene expression.