Two circular chromosomes of unequal copy number make up the mitochondrial genome of the rotifer Brachionus plicatilis

The monogonont rotifer Brachionus plicatilis is an emerging model system for a diverse array of questions in limnological ecosystem dynamics, the evolution of sexual recombination, cryptic speciation, and the phylogeny of basal metazoans. We sequenced the complete mitochondrial genome of B. plicatilis sensu strictu NH1L and found that it is composed of 2 circular chromosomes, designated mtDNA-I (11,153 bp) and mtDNA-II (12,672 bp). Hybridization to DNA isolated from mitochondria demonstrated that mtDNA-I is present at 4 times the copy number of mtDNA-II. The only nucleotide similarity between the 2 chromosomes is a 4.9-kbp region of 99.5% identity including a transfer RNA (tRNA) gene and an extensive noncoding region that contains putative D-loop and control sequence. The mtDNA-I chromosome encodes 4 proteins (ATP6, COB, NAD1, and NAD2), 13 tRNAs, and the large and small subunit ribosomal RNAs; mtDNA-II encodes 8 proteins (COX1-3, NAD3-6, and NAD4L) and 9 tRNAs. Gene order is not conserved between B. plicatilis and its closest relative with a sequenced mitochondrial genome, the acanthocephalan Leptorhynchoides thecatus, or other sequenced mitochondrial genomes. Polymerase chain reaction assays and Southern hybridization to DNA from 18 strains of Brachionus suggest that the 2-chromosome structure has been stable for millions of years. The novel organization of the B. plicatilis mitochondrial genome into 2 nearly equal chromosomes of 4-fold different copy number may provide insight into the evolution of metazoan mitochondria and the phylogenetics of rotifers and other basal animal phyla.     

Sterol regulatory element-binding proteins induce an entire pathway of cholesterol synthesis

To evaluate the effects of sterol regulatory element-binding proteins (SREBPs) on the expression of the individual enzymes in the cholesterol synthetic pathway, we examined expression of these genes in the livers from wild-type and transgenic mice overexpressing nuclear SREBP-1a or -2. As estimated by a Northern blot analysis, overexpression of nuclear SREBP-1a or -2 caused marked increases in mRNA levels of the whole battery of cholesterogenic genes. This SREBP activation covers not only rate-limiting enzymes such as HMG CoA synthase and reductase that have been well established as SREBP targets, but also all the enzyme genes in the cholesterol synthetic pathway tested here. The activated genes include mevalonate kinase, mevalonate pyrophosphate decarboxylase, isopentenyl phosphate isomerase, geranylgeranyl pyrophosphate synthase, farnesyl pyrophosphate synthase, squalene synthase, squalene epoxidase, lanosterol synthase, lanosterol demethylase, and 7-dehydro-cholesterol reductase. These results demonstrate that SREBPs activate every step of cholesterol synthetic pathway, contributing to an efficient cholesterol synthesis.     

Matrix mineralization as a trigger for osteocyte maturation.

The morphology of the osteocyte changes during the cell's lifetime. Shortly after becoming buried in the matrix, an osteocyte is plump with a rich rough endoplasmic reticulum and a well-developed Golgi complex. This "immature" osteocyte reduces its number of organelles to become a "mature" osteocyte when it comes to reside deeper in the bone matrix. We hypothesized that mineralization of the surrounding matrix is the trigger for osteocyte maturation. To verify this, we prevented mineralization of newly formed matrix by administration of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) and then examined the morphological changes in the osteocytes in rats. In the HEBP group, matrix mineralization was disturbed, but matrix formation was not affected. The osteocytes found in the unmineralized matrix were immature. Mature osteocytes were seen in the corresponding mineralized matrix in the control group. The immature osteocytes in the unmineralized matrix failed to show immunoreactivity with anti-sclerostin antibody, whereas mature osteocytes in the mineralized matrix showed immunoreactivity in both control and HEBP groups. These findings suggest that mineralization of the matrix surrounding the osteocyte is the trigger for cytodifferentiation from a plump immature form to a mature osteocyte. The osteocyte appears to start secreting sclerostin only after it matures in the mineralized bone matrix.     

Tenascin Expression in Vitro and in Vivo: Comparison between Epithelial and Nonepithelial Rat Cell Lines

Tenascin is a novel six-armed extracellular-matrix glycoprotein expressed in association with mesenchymal-epithelial interactions, and its expression is temporally and spatially restricted during organogenesis and carcinogenesis. The distribution and alterations in the expression of fibronectin, laminin, and especially of tenascin, were compared between in vitro and in vivo studies with rat epithelial (hepatocyte-derived) and nonepithelial (sarcoma-derived) cell lines. Immunoprecipitation studies revealed that the production of extracellular-matrix glycoproteins varied among the cell lines. Two ascites-hepatoma-derived cell lines and one sarcoma-derived line were found to synthesize tenascin in vitro. Their major tenascin isoform yielded a molecular weight of 220 kDa under reducing conditions. The other cell lines examined, including all of those derived from normal hepatocytes, were negative for the expression of tenascin. Coculture studies were performed between epithelial and nonepithelial cell lines. No drastic change in tenascin expression was found after coculturing the cells. As an in vivo study, cell lines were transplanted into nude mice. All xenografts of the epithelial lines were associated with a strong positive reaction for extracellular-matrix glycoproteins, and especially for tenasein, in the mouse fibrous stroma adjacent to them. This represents the epithelial induction of stromal tenascin. Whether or not they produced tenascin in vitro, after transplantation none of the epithelial cell lines themselves produced tenascin, whereas both of the nonepithelial cell lines prominently produced tenascin. These findings suggest that, in the process of interactions between epithelial and nonepithelial cells, the expression of tenascin depends on the switch from in vitro to in vivo.     

Ultrastructural evidence for a possible secretory function of Merkel cells in the barbels of a teleost fish,Cyprinus carpio

Summary Examination of barbels of the carp (Cyprinus carpio) revealed cells showing the characteristics of Merkel cells. Some ultrastructural features of these cells suggest a secretory function.     

Protein deficient chloroplast ribosomes in a yellow mutant of Chlamydomonas reinhardii

Ribosomes and ribosomal proteins from wild-type and a yellow mutant of Chlamydomonas reinhardii were analyzed and compared by two-dimensional gel electrophoresis. The mixothrophically grown yellow-76 mutant differs from wild-type cells in lowered chlorophyll content and photosynthetic activity per chlorophyll unit. The latter is connected with the decreased activity of the ribulose-I,5-diphosphate-carboxylase enzyme. Analytical ultracentrifugation of cell extracts shows a normal amount of free 70S ribosomes and 50S subunit in the mutant cells. Two-dimensional gel electrophoresis shows considerable alterations in the protein composition of the 70S ribosomes of the mutant. Two proteins are absent from the electrophoretograms of the yellow-76 mutant, and seven proteins are present in reduced amounts. The genetical analysis shows a Mendelian pattern of inheritance, indicating that protein alterations presumably are localized in nuclear DNA.Abbreviation MNNG N-methyl-N-nitro-N-nitrosoguanidine     

On the origin of plastids

The buoyant density in CsCl of ribosomes from chloroplasts of the green algaChlorella pyrenoidosa and two species of higher plants,Pisum sativum andChenopodium album, has been studied. From the relative protein content it was calculated that 70S ribosomes from chloroplasts are much smaller than 80S cytoplasmic ribosomes (3.0–3.1×10⁶ and 4.0×10⁶ daltons) and slightly larger than 70S ribosomes from abcteriaE. coli 2.5×10⁶ daltons). Chloroplast ribosomes from pea seedlings were analyzed by two-dimensional polyacrylamide gel electrophoresis. They appear to contain 71 proteins. This indicates that chloroplast ribosomes contain a larger number of proteins than do the ribosomes fromE. coli and other species of Enterobacteriaceae. Further study will permit a probable evaluation of the validity of Mereschkowsky's hypothesis that the photosynthetic plastids of eukaryotic plant cells are the evolutionary descendants of endosymbiotic blue-green algae.