Mutations in a Saccharomyces cerevisme host showing increased holding stability of the heterologous plasmid pSRI

Summary We have isolated Saccharomyces cerevisiae mutants, smp, showing stable maintenance of plasmid pSRI, a Zygosaccharomyces rouxii plasmid. The smp mutants were recessive and were classified into at least three different complementation groups. The three mutants also showed increased stability of YRp plasmids and the mutations are additive for plasmid stability. One mutation, smp1, confers a respiration-deficient (rho ⁰) phenotype and several Rho^– mutants independently isolated by ethidium bromide treatment of the same yeast strain also showed increased stabilities of pSR1 and YRp plasmids. The wild-type S. cerevisiae cells showed a strongly biased distribution of pSR1 molecules as well as YRp plasmids to the mother cells at mitosis, while the smpf mutant did not show this bias. Another mutation, smp3, at a locus linked to ade2 on chromosome XV, confers temperature-sensitive growth. The SMP3 gene encodes a 59.9 kDa hydrophobic protein and disruption of the gene is lethal.     

Immunofluorescence Microscopy of Microtubule Arrangement in Root Cells of Pisum sativum L. var Alaska

The arrangement of wall microtubules (MTs) in Pisum sativumroots was viewed immunofluorescently using cryosectioning. Mostcells in the tip region of pea roots (0–2 mm from tip)had wall MTs arranged transversely to the root axis. In theregion elongating at a higher rate (2–4 mm), wall MTsof epidermal, cortical and stelar cells were all transverselyarranged. In the region of about 5 mm from the tip, in whichcell elongation had already ceased, wall MTs in cortical cellschanged from a transverse to an oblique arrangement in relationto the root axis. Some cells had a crossed arrangement of wallMTs, which was interpreted as representing two sets of unidirectional,oblique wall MTs in opposite cell cortices of a single cell.This change was completed within a region of 1-mm width. Sinceroots elongated at a rate of 0.6 mm h^–1, it means thatthe arrangement of wall MTs changed within 2 h. An oblique arrangementof wall MTs was also observed in stelar cells. As the cellsaged, the oblique arrangement tended to change to a steeperor even a longitudinal one. (Received January 24, 1986; Accepted May 15, 1986)     

Molecular cloning and characterization of the Endo B cytokeratin expressed in preimplantation mouse embryos

A cDNA clone of a keratin-related, intermediate filament protein, designated Endo B, was constructed from size-fractionated parietal endodermal mRNA and characterized. The 1466-nucleotide cDNA insert contains an open reading frame of 1272 nucleotides that would result in 5' and 3' noncoding sequences of 54 and 60 nucleotides, respectively. The predicted amino acid composition, molecular weight (47,400), and peptide pattern correlate well with data obtained on the isolated protein. The predicted amino acid sequence fits easily into the general domain structure suggested for all intermediate filament proteins with a unique amino-terminal head domain, a large conserved central domain of predominantly alpha-helical structure, and a relatively unique carboxyl-terminal or tail domain. Over the entire molecule, Endo B is 43% identical with human 52-kDa epidermal type I keratin. However, over two of the three regions contained in the central domain that are predicted to form coiled-coil structures, the Endo B is 54-68% identical with other type I keratin sequences. This homology, along with the presence of the completely conserved sequence DNARLAADDFR-KYE, which is found in all type I keratins, permits the unambiguous identification of Endo B as a type I keratin. Comparison of the Endo B sequence to other intermediate filament proteins reveals 22 residues which are identical in all intermediate filament proteins regardless of whether filament formation requires only one type of protein subunit (vimentin, desmin, glial fibrillar acidic protein, or a neurofilament protein) or two dissimilar types (type I and type II keratins). Endo B mRNA was detectable in RNA isolated from F9 cells treated with retinoic acid for 48 h. Approximately three to five genes homologous to Endo B were detected in the mouse genome.     

Control of chromatophore movements in dermal chromatic units of blue damselfish--I. The melanophore

Mechanisms controlling pigment movements in the melanophore of the blue damselfish, Chrysiptera cyanea, were studied. Histological observations revealed that the melanophore had three-dimensionally developed processes to envelop overlying small iridophores, and thus participated in the construction of a simple dermal chromatophore unit. Nervous stimulation, catecholamines and melatonin brought about melanosome aggregation in the melanophore. The actions of the nervous stimulation and catecholamines were antagonized by alpha adrenolytic agents. A beta adrenergic agonist, metaproterenol, adenosine and adenine nucleotides, and alpha-MSH acted as pigment-dispersing agents. These results indicate that the melanophore of the present material is controlled quite orthodoxly by adrenergic nerves and endocrines, notwithstanding the fact that it has quite a unique morphology among fish species, and that its motile rate is remarkably high.     

Interaction of alpha-chymotrypsin with dimyristoyl phosphatidylcholine vesicles

The amidolytic activity of chymotrypsin for Suc-Ala2-Pro-Phe-MCA was somewhat enhanced by dimyristoyl PC at low ionic strength, but not at high ionic strength. The activity was strongly inhibited by pure egg yolk PA. The inhibition by 200 ng PA was neutralized by addition of 1 microgram dimyristoyl PC or pure egg yolk PC, which formed vesicles with the PA. The Km and kcat (s-1) values of chymotrypsin for hydrolysis of Suc-Ala2-Pro-Phe-MCA changed from 15 microM to 42 microM, 0.1 mM and 0.5 mM, and from 1.5 to 2.7, 3.7, and 1.0 in the presence of 1 microgram dimyristoyl PC, 0.5 micrograms pure egg yolk PE and 0.2 microgram egg yolk PA, respectively. Gel-filtration chromatography showed that dimyristoyl PC formed a complex with chymotrypsin, but did not interact with the substrate, indicating that the basic globular protein, chymotrypsin, interacted with net-neutral PL.     

Antigens associated with various cytotoxic activities of murine peripheral macrophages

BCG- or glucan-elicited murine peripheral macrophages released a cytotoxin in the presence of loach egg lectin, whereas proteose peptone-, glycogen-, or thioglycollate-elicited or resident macrophages did not. The macrophages that released cytotoxin coincided with those that showed lectin-dependent macrophage-mediated cytolysis (LDMC) in response to loach egg lectin. The cytotoxin released by BCG-elicited macrophages in response to loach egg lectin had a molecular weight of 55 K daltons. The macrophages that released cytotoxin and other cytotoxic macrophages such as those that showed LDMC- and antibody-dependent macrophage-mediated cytolysis (ADMC) were examined by using several antibodies to surface antigens of macrophages. The results showed that murine peripheral macrophages could be divided into three types. Resident macrophages (Type I) which had common macrophage antigens (Mac-1 and B12) showed only LDMC in response to wheat germ agglutinin. Some elicited macrophages (Type II) were asialo GM1-positive and showed both ADMC and LDMC in response to wheat germ agglutinin. Activated macrophages (Type III) showed LDMC in response to loach egg lectin and cytotoxin-release, but had no antigen detectable with monoclonal anti-macrophage antibody (C14). These three types of macrophages were clearly distinguished diagrammatically by their roof-shaped, rocket-shaped and irregular-shaped profiles of activities and antigens. These data suggest that several selected surface antigens of macrophages are associated with distinct cytotoxic stages of peripheral macrophages.     

Proton magnetic resonance studies of 7 Fe ferredoxins: Lower potential of the [4 Fe–4 S] redox center than the [3 Fe–3 S] cluster

Two types of iron-sulfur clusters, [3 Fe–3 S] and [4 Fe–4 S], were identified by ¹H-NMR in ferredoxins from Thermus thermophilus, Mycobacterium smegmatis and Pseudomonas ovalis. The [4 Fe–4 S] clusters always showed the redox couples which had potentials lower than that of the [3 Fe–3 S] clusters.     

Construction and Characterization of Isogenic Series of Saccharomyces cerevisiae Polyploid Strains

Tetraploid cells of Saccharomyces cerevisiae are generated spontaneously in a homothallic MATa/MATα diploid population at low frequency (approximately 10⁻⁶ per cell) through the homozygosity of mating-type alleles by mitotic recombination followed by homothallic switching of the mating-type alleles. To isolate tetraploid clones more effectively, a selection method was developed that used a dye plate containing 40 mg each of eosin Y and amaranth in synthetic nutrient agar per liter. It was possible to isolate tetraploid clones on the dye plate at a frequency of 1 to 3% among the colonies colored dark red in contrast to the light red of the original diploid colonies. Isogenic series of haploid to tetraploid clones with homozygous or heterozygous genomic configurations were easily constructed with the tetraploid strains. No significant differences in specific growth rate or fermentative rate were observed corresponding to differences in ploidy, although the haploid clones showed a higher frequency of spontaneous respiratory-deficient cells than did the others. However, a significant increment in the fermentative rate in glucose nutrient medium was observed in the hybrid strains constructed with two independent homozygous cell lines. These observations strongly suggest that the polyploid strains favored by the brewing and baking industries perform well not because of the physical increment of the cellular volume by polyploidy but because of the genetic complexity or heterosis by heterozygosity of the genome in the hybrid polyploid cells.     

Expression in Escherichia coli of chemically synthesized gene for the human immune interferon.

A 454 base pair fragment of double stranded DNA consisting of a gene for a human immune interferon (hIFN-gamma), initiation and termination signals plus appropriate restriction endonuclease sites, was totally synthesized. The synthesis involved preparation of 62 oligodeoxyribonucleotides by rapid, solid phase procedures, and enzymatic ligation of the oligonucleotides. This synthetic gene was expressed in E. coli under the control of the lac UV5 promoter. The product has antiviral activity which was acid labile and completely neutralized by antiserum to hIFN-gamma but not by antiserum to hIFN-alpha or hIFN-beta. Molecular weight of hIFN-gamma produced by E. coli was estimated to be about 32,000 and 17,000 by gel filtration and SDS-polyacrylamide gel electrophoresis respectively.