The Saccharomyces cerevisiae SOC8-1 gene and its relationship to a nucleotide kinase

The yeast SOC8-1 gene was originally identified by partial complementation of cdc8 mutant strains. We have carried out Bal31 deletion analysis of the SOC8-1 gene to define the minimal size which is required for the complementation of the cdc8 mutation. When the SOC8-1 gene is cloned in a multicopy plasmid, it enables temperature-resistant growth in the cdc8 mutant strain, while the SOC8-1 gene in a single copy plasmid does not. Thus, its suppression of the cdc8 mutant is dosage dependent. The high copy number vector carrying the SOC8-1 gene can complement five different cdc8 alleles, indicating that the suppression is not allele specific. Since CDC8 encodes thymidylate kinase, cells bearing a high copy number plasmid containing SOC8-1 gene were tested for the ability to phosphorylate several nucleoside monophosphates, including UMP, GMP and dTMP. Significantly increased phosphorylation activity was observed, suggesting that SOC8-1 encodes a nucleotide kinase. Both restriction enzyme analysis of the SOC8-1 gene and partial purification of the overproduced kinase in SOC8-1 overproducing strains suggest that SOC8-1 may be allelic with URA6. Consistent with these results, both SOC8-1 and URA6 are located on chromosome XI. Thus, one possible suppression mechanism is that SOC8-1 may provide a trans-acting dTMP kinase activity, bypassing the cdc8 gene defect.     

Immunoaffinity purification, partial sequence, and subcellular localization of rat liver phospholipase A2

Monoclonal antibodies against rat liver mitochondrial phospholipase A2 were used to develop a rapid immunoaffinity chromatography for enzyme purification. The purified enzyme showed a single band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the N-terminal 24 amino acids was determined. This part of the sequence showed only 25% homology with that of rat pancreatic phospholipase A2 but was 96% identical to that of rat platelet and rat spleen membrane-associated phospholipase A2. These enzymes are distinguished from pancreatic phospholipases A2 by the absence of Cys-11. In rat liver phospholipase A2 activity has been reported in various subcellular fractions. All of these require Ca2+ and have a pH optimum in the alkaline region, but little is known about the structural relationship and quantitative distribution of these enzymes. We have investigated these points after solubilization of the phospholipase A2 activity from total homogenates and crude subcellular fractions by extraction with 1 M potassium chloride. Essentially all of the homogenate activity could be solubilized by this procedure indicating that the enzymes occurred in soluble or peripherally membrane-associated form. Gel filtration and immunological cross-reactivity studies indicated that phospholipases A2 solubilized from membrane fractions shared a common epitope with the mitochondrial enzyme. The quantitative distribution of the immunopurified enzyme activity among subcellular fractions followed closely that of the mitochondrial marker cytochrome c oxidase. Rat liver cytosol contained additional Ca2+-dependent and -independent phospholipase activities.     

Conformational feature of neuroactive domoic acid: X-ray structural comparison with isodomoic acid A and alpha-kainic acid.

As an aid for developing a new type of potent insecticide acting on the neuromuscular junction, conformational characteristics of domoic acid and isodomoic acid A, the naturally occurring glutamate agonists, were investigated by X-ray crystal analyses. Conformational comparison with a neuroactive alpha-kainic acid provides information concerning the stereochemical feature responsible for the biological activity.     

Regulation of expression of a wound-inducible tomato inhibitor I gene in transgenic nightshade plants

A wound-inducible proteinase Inhibitor I gene from tomato containing 725 bp of the 5 region and 2.5 kbp of the 3 region was stably incorporated into the genome of black nightshade plants (Solanum nigrum) using an Agrobacterium Ti plasmid-derived vector. Transgenic nightshade plants were selected that expressed the tomato Inhibitor I protein in leaf tissue. The leaves of the plants contained constitutive levels of the inhibitor protein of up to 60 g/g tissue. These levels increased by a factor of about two in response to severe wounding. Only leaves and petioles exhibited the presence of the inhibitor, indicating that the gene exhibited the same tissue specificity of expression found in situ in wounded tomato leaves. Inhibitor I was extracted from leaves of wounded transformed nightshade plants and was partially purified by affinity chromatography on a chymotrypsin-Sepharose column. The affinity-purified protein was identical to the native tomato Inhibitor I in its immunological reactivity and in its inhibitory activity against chymotrypsin. The protein exhibited the same M _r of 8 kDa as the native tomato Inhibitor I and its N-terminal amino acid sequence was identical to that of the native tomato inhibitor I, indicating that the protein was properly processed in nightshade plants. These expriments are the first report of the expression of a member of the wound-inducible tomato Inhibitor I gene family in transgenic plants. The results demonstrate that the gene contains elements that can be regulated in a wound-inducible, tissuespecific manner in nightshade plants.     

Xylanase prebleaching of fractions of Douglas-fir kraft pulp of different fibre length

A commercial xylanase from Trichoderma longibrachiatum was used to treat the fractions of Douglas-fir kraft pulp of different fibre length. Enzymatic prebleaching was followed by chelation and peroxide bleaching. An evaluation of both optical and physical properties of the distinct fractions was conducted. A difference in susceptibility of the fractions of different fibre length to xylanase prebleaching was observed. The bleach-boosting effect observed with all fractions appeared to be related to the high-molecular-mass UV-absorbing material solubilized during enzyme treatments. Xylanase treatments resulted in beneficial effects to handsheet density and roughness as well as to some of the strength properties. However, the response to the xylanase treatments exhibited by all fractions of different fibre length was not uniform, indicating that fiber composition played a key role in the effectiveness of xylanase treatments.     

Selective Loss of Sperm Bearing a Compound Chromosome in the Drosophila Female

The Drosophila compound entire second chromosome, C(2)EN, displays paternal transmission well below Mendelian expectations (NOVITSKI et al. 1981). Because C(2)EN stocks also show higher-than-expected rates of zygotic lethality, it was proposed that this reduced paternal inheritance might be wholly or partially due to postfertilization events. Efforts to investigate this phenomenon have been hampered because the progeny of crosses between C(2)EN-bearing individuals and those with normal karyotypes die during embryogenesis. We have circumvented this obstacle by employing fluorescence in situ hybridization to directly karyotype early embryos from crosses involving C(2)EN-bearing individuals. This analysis reveals that the distortion in paternal transmission is established before fertilization. Moreover, measurement of the sperm ratios within both the male and female reproductive organs demonstrates that C(2)EN-bearing sperm are selectively lost after sperm transfer to the female and before storage of sperm in the seminal receptacles and spermathecae. Our results are consistent with a model of meiotic drive in which aberrations occuring early in meiosis lead ultimately to sperm dysfunction.     

An Integrated Map with Cosmid/PAC Contigs of a 4-Mb Down Syndrome Critical Region

The major phenotypic features of Down syndrome have been correlated with partial trisomies of chromosome 21, allowing us to define the candidate gene region to a 4-Mb segment on the 21q22.2 band. We present here a high-resolution physical map with megabase-sized cosmid/PAC contigs. This ordered clone library has provided unique material for the integration of a variety of mappable objects, including exons, cDNAs, restriction sites, etc. Furthermore, our results have exemplified a strategy for the completion of the chromosome 21 map to sequencing.     

Transient reduction in secreted 32 kD chitinase prevents somatic embryogenesis in the carrot (Daucus carota L.) variant ts11

At the nonpermissive temperature, somatic embryos of the temperature-sensitive (ts) carrot (Daucus carota L.) cell variant ts11 only proceed beyond the globular embryo stage in the presence of medium conditioned by wild-type cells. The causative component in the conditioned medium has been identified as an acidic 32 kD endochitinase. An antiserum raised against the 32 kD chitinase detected this protein in culture medium from ts11 embryo cultures grown at the permissive temperature as well as at the nonpermissive temperature. No difference in biochemical characteristics or in effect on ts11 embryo development could be detected between the 32 kD chitinase purified from wild-type cultures and the chitinase from ts11 cultures grown at the permissive or at the nonpermissive temperature. Compared to the amount present in a ts11 embryo culture at the permissive temperature, a reduction in the amount of 32 kD chitinase was observed during the temperature-sensitive period at the nonpermissive temperature. These results imply that the arrested embryo phenotype of ts11 is not the result of a structural difference in its 32 kD chitinase, but is the result of a transient decrease in the amount of 32 kD chitinase present. Morphological observations indicate that the ts11 phenotype is pleiotropic and also affects the cell wall of nonembryogenic cells. © 1995 Wiley-Liss, Inc.     

Production of halogenated compounds byBjerkandera adusta

The white-rot fungusBjerkandera adusta produces volatile chlorinated phenyl compounds. The main compounds identified were 3-chloro-4-methoxybenzaldehyde (3-chloro-p-anisaldehyde), 3-chloro-4-methoxybenzyl alcohol (3-chloro-p-anisyl alcohol), 3,5-dichloro-4-methoxybenzaldehyde (3,5-dichloro-p-anisaldehyde), and 3,5-dichloro, 4-methoxybenzyl alcohol (3,5-dichloro-p-anisyl alcohol).p-Anisaldehyde, veratraldehyde and the corresponding alcohols,p-anisyl alcohol and veratryl alcohol were produced simultaneously. Even with a very low concentration of chloride in the medium (< 10^–5 m), chlorinated aromatic compounds were still observed. Addition of bromide to the culture medium led to the production of brominated compounds: 3-bromo-4-methoxybenzaldehyde, 3-bromo-4-methoxybenzyl alcohol, 3,5-dibromo-4-methoxybenzaldehyde and 3-bromo-5-chloro-4-methoxybenzaldehyde. These brominated compounds have not previously been reported as natural products. Although iodo-aromatic compounds were not produced by supplementation of the medium with iodide, isovanillin was found in the culture broth under these conditions. This compound may be formed by substitution of the iodine intermediate by a hydroxyl group on the third carbon of the ring. Diiodomethane or chloroiodomethane were also found. It is the first time that the production of halomethane has been related to the production of halogenated aromatic compounds. All the strains tested have these capabilities.