Phospholipid requirements of membrane-bound chitin synthase from Absidia glauca

Abstract Membrane-bound chitin synthase, a key enzyme in chitin biosynthesis, had a specific requirement for phospholipid. The activity of the enzyme was enhanced 2.7-fold by adding phosphatidylinositol from porcine liver but not by other phospholipids. Each of the constituents of phospholipids inhibited enzyme activity at concentrations over 0.05%. Sterols and glycolipids had little effect on chitin synthase activation. Moreover, investigation using define species of phosphatidylcholine revealed that 1-palmytoyl-2-arachidoyl and 1-stearoyl-2-arachidoyl phosphatidylcholine activated the enzyme. In contrast to the arachidoyl acyl chain, other species having unsaturated fatty acyl chains inhibited enzyme activity at a concentration of 0.01%.     

Conservation and variation of ribosomal DNA in eukaryotes

The organization of ribosomal DNA clusters, repetitive units, and their conserved and variable elements in eukaryotes is considered with special emphasis on the structure and function of the ribosomal intergenic spacer (rIGS). Data on rDNA variation, evolution, and possible role in various biological processes are reviewed.     

Conservation of ribosomal RNA gene arrangement in the mitochondrial DNA of angiosperms

In six different angiosperms, mitochondrial genes for 18S and 5S rRNAs were found to be closely linked but distant from mitochondrial 26S rRNA genes.This is paper no. 5 in the series Organization and Expression of the Mitochondrial Genome of Plants     

Variable arrangement of 5S ribosomal genes within the ribosomal DNA repeats of arthropods.

The polymerase chain reaction and hybridization to genomic blots were used to investigate whether the previously observed inclusion of 5S ribosomal RNA genes in the 28S-18S ribosomal DNA intergenic regions of some crustacean species (copepods) could also be detected in other arthropods. Such an arrangement was found not only in other calanoid copepod species but also in a cirriped, an euphausid, and a spider. It is interesting that species from two different calanoid copepod genera do not have this type of arrangement. We conclude that the inclusion of 5S ribosomal RNA genes within the ribosomal DNA repeats has probably occurred repeatedly during the evolution of arthropod species and that the mechanism(s) responsible for these insertions could also be responsible for their loss.     

The SEG1 element: a new DNA region promoting stable mitotic segregation of plasmids in the zygomycete Absidia glauca.

Summary A series of new vectors for the model zygomycete Absidia glauca was constructed on the basis of the structural neomycin resistance (Neo^r) gene controlled by the promoter of the gene for elongation factor 1 (TEF). In order to select for transformed colonies with a stable Neo^r phenotype, spores from primary transformants were pooled and grown for two sporulation cycles under non-selective conditions. Southern blot analysis of DNA from single spore isolates originating from independent transformant pools allowed the identification of two autonomously replicating plasmids. Retransformation of Escherichia coli and restriction analysis of the two plasmids provided evidence for spontaneous in vivo insertion of a new DNA element (SEG1) from the A. glauca genome. The inserted regions in both plasmids are essentially identical and do not represent repetitive DNA. Compared with other autonomously replicating vectors, these SEG1-containing plasmids are mitotically extremely stable and are passed on to the vegetative spore progeny of a retransformed A. glauca strain. We assume that SEG1 contains structural elements involved in partitioning and stable segregation of plasmids. For the construction of stable transformants of A. glauca, the SEG1 element may be regarded as a major breakthrough, because stabilization of transformed genetic traits by integration is difficult to achieve in all mucoraceous fungi and all known replicating plasmids are mitotically unstable.     

Spatial pattern of chloroplast DNA variation of Cyclobalanopsis glauca in Taiwan and East Asia

This study examined the spatial pattern of chloroplast DNA (cpDNA) variation in Cyclobalanopsis glauca (Thunb. ex Murray) Oerst. (Fagaceae) in 140 trees from Taiwan (25 populations), Japan (three), Ryukyus (two), Hong Kong (one) and Mainland China (one). By sequencing three cpDNA intergenic spacer fragments using universal primers (trnT-trnL, trnV-trnM, including the trnV intron, and petG-trnP), we found a total of 1,980 bp and 15 polymorphic sites. Among them, 12 sites were caused by point mutation, and three resulted from insertion. This gives rise to a total of 13 cpDNA haplotypes. The level of differentiation among the populations studied is relatively high (GST = 0.612). Two ancestral haplotypes (A and B) are distributed widely in East Asia. Interestingly, all the derived cpDNA variations are found only in Taiwan but not in other areas. The Central Mountain Ridge (CMR) of Taiwan creates an unsurpassed barrier to the east-west gene flow of C. glauca. Among the populations on the west of CMR, only three separated populations, Yangmingshan, Wushe and Chinshuiying, have high haplotype diversity, each consisting of sister haplotypes all mutated from the same ancestral haplotype. Thus, they have probably originated from de novo mutation after the last glaciation. This inference agrees with the observation that no spatial autocorrelation existed on the west side. Two unrelated dominant lineages on the east of the CMR (haplotypes D and F) showed significant spatial genetic structure. Estimate of NST - GST was -0.090 and differed significantly from zero. Thus at the local scale, the phylogeographical component of the genetic structure is significant on the east of the CMR. Accompanied by published palynological records of the last glaciation, this study suggests the possibility that these two types were colonized northward from the southeastern part of Taiwan. 'Star-like' genealogy is characterized, with all the haplotypes coalescing rapidly and as a general outcome of population expansion (Page & Holmes 1998). A neutrality test also suggested a demographic expansion recovered from a bottleneck. We therefore inferred that the southeastern part of Taiwan might be a potential refugium for C. glauca.     

Green fluorescent protein as a reporter for gene expression in the mucoralean fungus Absidia glauca

Mucoralean fungi (Zygomycota) are used for many industrial processes and also as important model organisms for investigating basic biological problems. Their genetic analysis is severely hampered by low transformation frequencies, by their strong tendency towards autonomous replication of plasmids instead of stable integration, and by the lack of reliable genetic reporter systems. We constructed plasmids for transforming the model zygomycete Absidia glauca that carry the versatile reporter gene coding for green fluorescent protein (GFP). gfp expression is controlled either by the homologous actin promoter or the promoter for the elongation factor of translation, EF1alpha. These plasmids also confer neomycin resistance and carry one of two genetic elements (rag1, seg1) that improve mitotic stability of the plasmid. The gfp constructs were replicated extrachromosomally and could be recovered from retransformed Escherichia coli cells. gfp expression was monitored by epifluorescence microscopy. The gfp reporter gene plasmids presented here for the model zygomycete A. glauca constitute the first reliable system that allows the monitoring of gene expression in this important group of fungi.     

Comparative analysis of intra-individual and inter-species DNA sequence variation in salmonid ribosomal DNA cistrons

This study examines sequence divergence in three spacer regions of the ribosomal DNA (rDNA) cistron, to test the hypothesis of unequal mutation rates. Portions of two transcribed spacers (ITS-1 and 5' ETS) and the non-transcribed spacer (NTS) or intergenic spacer (IGS) formed the basis of comparative analyses. Sequence divergence was measured both within an individual lake trout (Salvelinus namaycush) and among several related salmonid species (lake trout; brook trout, Salvelinus fontinalis; Arctic char, Salvelinus alpinus; Atlantic salmon, Salmo salar; and brown trout, Salmo trutta). Despite major differences in the length of the rDNA cistron within individual lake trout, minimal sequence difference was detected among cistrons. Interspecies comparisons found that molecular variation in the rDNA spacers did not conform to the predicted pattern of evolution (ITS spacers相似文献   

Microprojectile bombardment as a reliable method for transformation of the mucoralean fungus Absidia glauca

Genetic analysis of all Mucor-like fungi is severely impaired by the low efficiency of transformation systems and the genetic instability of the introduced plasmid constructs. The transformation efficiency of one of the model systems among mucoralean fungi, Absidia glauca, was improved considerably by microprojectile bombardment. For this purpose, a plasmid was constructed conferring (i) neomycin resistance as a selective marker and (ii) fluorescence due to expression of the gfp gene from the jellyfish Aequorea victoria. Compared with previous techniques, this method offers increased efficiency, with considerably easier handling than procedures based on protoplasts and, therefore, improved reliability. The uninucleate sporangiospores of A. glauca can be transformed early during the germination process. At this stage the number of nuclei ranges between 1 and 2. Thus, the abundance of transgenic nuclei in the coenocytic mycelia is high, and fewer problems are encountered with detecting low expression levels of the genes used for selection and monitoring of transformants.     

D-Mannitol dehydrogenase from Absidia glauca. Purification, metabolic role, and subunit interactions.

When Absidia glauca was grown in minimal media with D-mannitol as the only source of carbon, an NAD+ specific D-mannitol dehydrogenase (EC 1.1.1.67) was induced. The crude extract also gave evidence of mannitol kinase, mannitol-1-phosphate dehydrogenase, phosphofructokinase, and L-iditol dehydrogenase activity. The heat labile purified preparation was judged enzymically homogeneous based on evidence derived from substrate specificity studies and activity staining, following disc gel electrophoresis. The enzymic monomer, with a weight of about 67000 daltons, slowly polymerizes when stored at -20 degrees C, giving a multiplicity of protein bands on electrophoresis distributed predominantly across a spectrum from dimer to pentamer, with enzymic activity resident predominantly in even multiples of the monomer. Depolymerization occurred rapidly (hours) when a frozen preparation was brought to and held between 4 and 20 degrees C. Aggregate fragmentation with sodium dodecyl sulfate showed a time-temperature dependence, terminating in a subunit component of 13000 daltons. pH optimum for polyol oxidation occurs at 9.6 (NaOH-glycine buffer) while ketose reduction proceeded most rapidly at pH 7.0-7.2 (phosphate buffer). A regulatory role is suggested for this enzyme based on dead-end inhibition by mannitol 1-phosphate, multiple enzyme forms, and its locus at the initiation site for mannitol utilization. The physiological relevance of low-temperature aggregation to regulatory control remains to be established.     

A molecular basis for discrete size variation in human ribosomal DNA.

The tandemly repeated human ribosomal RNA (rRNA) genes contain a region of size heterogeneity that is present in the nontranscribed spacer of every individual examined. This heterogeneity has been previously examined by Southern analysis of BamHI-digested human DNA. Using a ribosomal DNA (rDNA) probe specific for the 3' end of the 28S rRNA gene, at least four discrete sizes of BamHI fragments were seen in human populations. Molecular analysis of the cloned DNA from this region reveals tandem duplication of a segment of spacer rDNA located 388 base pairs (bp) 3' to the end of the 28S ribosomal RNA gene. Five hundred fifty bp of DNA, flanked on either side by a 150-bp repeated element, is either duplicated or deleted to produce a series of spacers that differ in size by 850 bp. These duplications/deletions appear to be the product of unequal homologous exchange, mediated by the small repeated element. Thus, human rDNA fragments cloned in lambda vectors and propagated in E. coli generate the same apparent size variation seen in genomic DNA. This study suggests that unequal homologous exchange is the molecular basis for the observed length heterogeneity in the spacer rDNA and may be a common mechanism for the generation of human genetic diversity.     

The arrangement of length heterogeneity in repeating units of amplified and chromosomal ribosomal DNA from Xenopus laevis

Non-transcribed spacer regions of Xenopus laevis ribosomal DNA have been found which vary in length between 1.8 × 10⁶ and 5.5 × 10⁶ daltons. Length variation of rDNA² repeats exists within a single nucleolar organizer. Amplified rDNA contains repeats of the same size classes but often in different abundance than the chromosomal rDNA of the same animal. If a certain repeat length is preferred during amplification in an individual, it is also preferred in siblings with the same chromosomal rDNA composition. Thus, preference for a size class in amplification is inherited. Some animals selectively amplify repeat lengths which are rarely found in their chromosomal rDNA; others amplify their most abundant size class.The intramolecular arrangement of length variability was analyzed by the electron microscopy of heteroduplex molecules. Long single strands from two separate preparations of amplified and chromosomal rDNA each were reannealed with an homogeneous cloned spacer-containing rDNA fragment (CD30), and the size of adjacent heteroduplex regions was determined. The arrangement of length heterogeneity is very different in the two types of rDNA. Most, if not all, tandem repeats along a single molecule of amplified rDNA are equal in length. This observation supports a rolling circle mechanism for amplification. In contrast, between 50% and 68% of adjacent repeats in a given molecule of chromosomal rDNA differ in length. For one of the chromosomal rDNA preparations analyzed, the frequency of non-identical nearest-neighbors is compatible with random scrambling of repeats of different lengths. This result bears on the mechanism by which tandem genes evolve. It rules out sudden correction mechanisms of tandem genes such as the “master-slave” or certain “expansion-contraction” models, which predict that tandem genes will be identical.     

华东地区青冈种群的等位酶变异

华东地区６个青岗种群等位酶变异的检测结果表明,青冈种群的遗传变异较大,每位点含有的等位基因较多,平均为２．３个,种水平的平均每位点等位基因数目为２．４。种群水平有效等位基因数目为１．４４６,种水平为１．４６７。Ｈａｒｄｙ－Ｗｅｉｎｂｅｒｇ平衡检验表明,青冈种群中不少位点偏离该平衡,其原因是种群内含有较多的纯合子,表现为多数位点的固定指数大于０,其中有９个位点的固定指数与０的偏差达到显著水平。     

Circular ribosomal DNA and ribosomal DNA: replication in somatic amphibian cells

Pulse labelled rDNA from cultured somatic cells of Xenopus laevis was examined by electron microscope autoradiography. The pattern of replication closely resembles that of bulk chromosomal DNA and differs considerably from rDNA synthesis during amplification in the oocyte. - About 0.15% of the rDNA molecules in the purified preparations were circular. The presence of interlocked circles of equal size indicates that the circles are not in vitro cyclization artefacts, but may represent free rRNA genes. A low frequency of circles was also seen in Xenopus blood rDNA. Their stability in high concentration of formamide suggests that they too did not arise after DNA extraction.     

Seasonal variation in the tissue water relations of Picea glauca

Seasonal variation in water relations of 3-yearold white spruce (Picea glauca (Moench) Voss) shoots, monitored with pressure-volume curves over 28 months, was closely related to shoot phenology and was sensitive to environmental fluctuations during both summer growth and winter dormancy. Turgor maintenance capacity was lowest during rapid shoot elongation from late May to early July; this was indicated by the lowest total turgor pressures, the highest (least negative) osmotic potentials at full turgor and the turgor loss point, the smallest differences between osmotic potentials at full turgor and the turgor loss point, the highest relative water contents at turgor loss and a linear decline in cell elasticity with decreasing turgor pressure. This suggests that the high susceptibility of white spruce seedlings to growth check after transplanting is largely attributable to the poor turgor maintenance capacity of this species in early summer.