A simple and rapid method for screening bacteria for type II restriction endonucleases: enzymes in Aphanothece halophytica

A method is described which allows a large number of bacterial strains to be rapidly and easily screened for the presence of site-specific endonucleases. The method involves selective permeabilization of the bacterial cell and analysis of the exuded material. Type II restriction endonucleases from cyanobacteria and Gram-negative eubacteria have been detected and new enzymes have been found. The method should be widely applicable and easy to modify for use in genera other than those tested. Three-site-specific endonuclease activities, detected by this method in Aphanothece halophytica PCC 7412, were purified and their recognition and cleavage specificities were determined AhaI and AhaII recognise and cleave the same DNA sequences as CauII and AcyI respectively; the specificity of AhaIII (TTTAAA) has been reported previously (Whitehead and Brown, 1982, FEBS Letters 143:296–300).Abbreviations Brij-58 20 cetyl ether - Pu purine nucleoside - Py pyrimidine nucleoside     

DNA probe localization at 18p113 band by in situ hybridization and identification of a small supernumerary chromosome

Summary Recombinant plasmid clone B74 (also named D18S3) containing a human single-copy DNA segment of 6 kilobases (kb) was localized by in situ hybridization on band p113 of chromosome 18. This probe was then used in cytogenetic diagnosis to identify precisely a small supernumerary chromosome as an isochromosome i(18p).     

Differential hormonal action of the bag cell neurons on the arterial system ofAplysia

Summary The peptide-secreting bag cell neurons ofAplysia californica activate a long-lasting, complex behavior called egg laying. During egg laying some organ systems (reproductive) are more active than others (digestive) suggesting that blood flow to these tissues may change in accordance with their activities during egg laying. To examine this possibility we used a semi-intact preparation of the three major arteries innervated by the abdominal ganglion. We found that electrically stimulated bursts of bag cell activity triggered a long-lasting (>1 h) increase in contractile activity in two arteries, the anterior and gastroesophageal, but did not affect contractions of the third (abdominal) artery. The arterial responses were not affected either in form or duration by denervation of the arteries, suggesting that the increase in contractile activity was mediated by hormonal actions of bag cell transmitters on vasoconstrictor muscles. In intact animals this differential action on the arterial system may cause a long-term decrease in blood flow to relatively inactive tissues (digestive and locomotory organs) while increasing circulation to tissues involved in egg production (ovotestis and oviduct).Abbreviations ASW artificial sea water - BCA bag cell activation - ELH egg laying hormone     

An Autosomal Gene That Affects X Chromosome Expression and Sex Determination in CAENORHABDITIS ELEGANS

Recessive mutant alleles at the autosomal dpy-21 locus of C. elegans cause a dumpy phenotype in XX animals but not in XO animals. This dumpy phenotype is characteristic of X chromosome aneuploids with higher than normal X to autosome ratios and is proposed to result from overexpression of X-linked genes. We have isolated a new dpy-21 allele that also causes partial hermaphroditization of XO males, without causing the dumpy phenotype. All dpy-21 alleles show hermaphroditization effects in XO males that carry a duplication of part of the X chromosome and also partially suppress a transformer (tra-1) mutation that converts XX animals into males. Experiments with a set of X chromosome duplications show that the defects of dpy-21 mutants can result from interaction with several different regions of the X chromosome. We propose that dpy-21 regulates X chromosome expression and may be involved in interpreting X chromosome dose for the developmental decisions of both sex determination and dosage compensation.     

Lectin-induced modulation of the antibody response to type III pneumococcal polysaccharide

Several lectins were tested for their capacity to alter the antibody response to type III pneumococcal polysaccharide (SSS-III). The antibody response was enhanced by concanavalin A (Con A), phytohemagglutinin (PHA), as well as lectins from Phytolacca americana (Pa-2), Pisum sativum (PSA), and Lens culinaris (LCH), when these lectins were given 2 days after immunization with SSS-III; however, suppression was obtained when Con A and Pa-2 were given at the time of immunization. By contrast the lectins from Vicia villosa (VVL) and Bauhinia purpurea (BPA) did not alter the antibody response. Since the lectins PSA and LCH bind to the same monosaccharide as Con A, whereas the other lectins bind to different monosaccharides, these findings indicate that there is no relationship between nominal monosaccharide specificity and the capacity to modulate the antibody response. Substantial increases in the magnitude of the IgG₁ antibody response was noted after the administration of Con A whereas profound enhancement of IgG_2a antibody response was noted after PHA was given.     

Location of O-acetyl groups in the acidic d-xylan of mimosa scabrella (bracatinga). A study of O-acetyl group migration

Extraction with dimethyl sulfoxide of wood-meal of the stem of bracatinga (Mimosa scabrella), a south Brazilian hardwood, that was defatted and delignified by treatment with aqueous chlorine at 0–5° followed by extraction with cold ethanol, gave a soluble O-acetylated 4-O-methyl-d-glucurono-d-xylan having (1→4)-linked β-d-xylopyranosyl residues that were unsubstituted (65%) and 2-O-(14%), 3-O- (16%), and 2,3-di-O-acetylated (5%), as determined by methylation analysis. Another preparation obtained by use of refluxing ethanol in the delignification process showed neither removal nor migration of acetyl groups. By comparison with synthetic, partly O-acetylated d-xylans of known composition, ¹³C-n.m.r. spectroscopy indicated that O-acetyl group migration does not occur during treatment with cold aqueous chlorine, refluxing ethanol, or water at 70°. Methyl 2-O-acetyl-4-O-methyl-β-d-xylopyranoside (6) was also unaffected by aqueous chlorine. O-Acetyl group migration took place more readily in aqueous and dimethyl sulfoxide solutions of 6 than of O-acetyl-d-xylans. The lowest temperatures at which migration was observed in monosaccharides was at 50 and 70° for solutions in D₂O and (CD₃)₂SO, respectively.     

Nonvisual feeding in a visual planktivore,Xenomelaniris venezuelae

Summary Studies of the diel feeding patterns of the planktivorous fish, Xenomelaniris venezuelae, in Lake Valencia, Venezuela, revealed that, although the fish is primarily a diurnal feeder, it consumes substantial numbers of Chaoborus larvae and pupae at night. A number of fish species are known which feed on plankton at night, but these fish are filter feeders and their diets largely consist of relatively small, nonevasive prey. Chaoborus, however, is large and agile. Predation by Xenomelaniris in the dark was also studied experimentally. Captured fish were placed in completely darkened aquaria with zooplankton from Lake Valencia. After several hours the plankton was removed and examined for evidence of feeding. The fish were found to consume Chaoborus pupae and fourth instar larvae but not other types of prey. The mode of feeding by Xenomelaniris in the dark is unknown.     

Phospholipid-Sensitive Ca2+-Dependent Protein Kinase Preferentially Phosphorylates Serine-115 of Bovine Myelin Basic Protein

Phospholipid-sensitive Ca2+ -dependent protein kinase (PL-Ca-PK) and cyclic AMP-dependent protein kinase (A-PK) both preferentially phosphorylated serine residues of bovine myelin basic protein (MBP). Tryptic peptide maps of MBP phosphorylated by PL-Ca-PK or A-PK, however, revealed different phosphopeptides, suggesting a difference in the intramolecular substrate specificity for the two enzymes. Serine-115 of MBP, in the sequence (-Arg-Phe-Ser(115)-Trp-), was found to be a preferred and probably major phosphorylation site for PL-Ca-PK. Because serine-115 of bovine MBP corresponds to serine-113 of rabbit MBP, an in vivo phosphorylation site reported by Martenson et al. (1983), and PL-Ca-PK is present at a very high level in brain and myelin, it is suggested that the enzyme may be responsible for the in vivo phosphorylation of this and other sites in MBP.     

A novel method for the rapid cloning in Escherichia coli of Bacillus subtilis chromosomal DNA adjacent to Tn917 insertions

Summary A rapid and general procedure has been devised for the pBR322-mediated cloning in Escherichia coli of Bacillus subtilis chromosomal DNA extending in a specified direction from any Tn917 insertion. Derivatives of Tn917 have been constructed that contain a pBR322-derived replicon, together with a chloramphenicol-resistance (Cm^r) gene of Gram-positive origin (selectable in B. subtilis), inserted by ligation in two orientations into a SalI restriction site located near the center of the transposon. When linearized plasmid DNA carrying such derivatives was used to transform to Cm^r B. subtilis bacteria already containing a chromosomal insertion of Tn917, the pBR322 sequences efficiently became integrated into the chromosomal copy of the transposon by homologous recombination. It was then possible to clone chromosomal sequences adjacent to either transposon insertion junction into E. coli, using a selection for ampicillin-resistance, by transforming CaCl₂-treated cells with small amounts of insert-containing DNA that had been digested with various restriction enzymes and then ligated at a dilute concentration. Because pBR322 sequences may be inserted by recombination in either orientation with respect to the transposon arms, a single restriction enzyme (such as EcoRi or SphI) that has a unique recognition site in pBR322 DNA may be used to separately clone chromosomal DNA extending in either direction from the site of any transposon insertion. A family of clones generated from the region of an insertional spo mutation (spoIIH::Tn917) was used in Southern hybridization experiments to verify that cloned material isolated with this procedure accurately reflected the arrangement of sequences present in the chromosome. Strategies are discussed for taking advantage of certain properties inherent in the structure of clones generated in this way to facilitate the identification and study of promoters of insertionally mutated genes.     

Physical mechanisms by which low-frequency magnetic fields can affect the distribution of counterions on cylindrical biological cell surfaces

Effects of dc and low-frequency ac magnetic fields on the motion and distribution of counterions on surfaces of cylindrical biological cells are examined. Magnetic fields along the cell axis as well as perpendicular to it are considered. When a dc magnetic field of any physically realizable magnitude is parallel to the cell axis it has no effect on ion motion, since the resulting Lorentz force is much smaller than the counterion-to-ion attractive force. However a dc magnetic field perpendicular to the cell surface will distort any preexisting ion motion and the resulting current (i) perpendicular to the original motion will be much larger than any current induced by a low-frequency ac magnetic field of the same magnitude as the dc field and parallel to it. Nevertheless i will still be much smaller than the current i_o constituting the original ion motion since (i/i_o)=/, where is the ion cyclotron frequency and the effective counterion collision frequency. With no preexisting coherent ion motion (i_o=0) the circulating current induced by a sinusoidally time-varying magnetic field parallel to the cell axis will be well below thermal fluctuation noise as long as only a single cell is considered; however when even an infinitesimal exchange of ions between adjacent cells is assumed the magnetic field will cause a redistribution of counterions on the cell surface. The resulting steady-state distribution becomes independent of the frequency of the applied magnetic field () when , where is 1/2 of the relaxation frequency for counterion diffusion. On the basis of these results it is suggested that whenever modification of cell behavior in response to application of a low-frequency magnetic field is established, measurements of dielectric permittivity versus frequency of the cell preparation be performed. Redistribution of counterions on the cell surface would be a likely cause if the noted effect becomes independent of the frequency of the applied magnetic field above the counterion dielectric relaxation frequency. It is also suggested that in magnetic field exposure of cell preparations the size of the sample (e.g. diameter of Petri dish) and direction of the magnetic field relative to average cell orientation can critically affect experimental results.