The WW domain protein PRO40 is required for fungal fertility and associates with Woronin bodies

Fruiting body formation in ascomycetes is a highly complex process that is under polygenic control and is a fundamental part of the fungal sexual life cycle. However, the molecular determinants regulating this cellular process are largely unknown. Here we show that the sterile pro40 mutant is defective in a 120-kDa WW domain protein that plays a pivotal role in fruiting body maturation of the homothallic ascomycete Sordaria macrospora. Although WW domains occur in many eukaryotic proteins, homologs of PRO40 are present only in filamentous ascomycetes. Complementation analysis with different pro40 mutant strains, using full-sized or truncated versions of the wild-type pro40 gene, revealed that the C terminus of PRO40 is crucial for restoring the fertile phenotype. Using differential centrifugation and protease protection assays, we determined that a PRO40-FLAG fusion protein is located within organelles. Further microscopic investigations of fusion proteins with DsRed or green fluorescent protein polypeptides showed a colocalization of PRO40 with HEX-1, a Woronin body-specific protein. However, the integrity of Woronin bodies is not affected in mutant strains of S. macrospora and Neurospora crassa, as shown by fluorescence microscopy, sedimentation, and immunoblot analyses. We discuss the function of PRO40 in fruiting body formation.     

Matrilin-3 is dispensable for mouse skeletal growth and development

Matrilin-3 belongs to the matrilin family of extracellular matrix (ECM) proteins and is primarily expressed in cartilage. Mutations in the gene encoding human matrilin-3 (MATN-3) lead to autosomal dominant skeletal disorders, such as multiple epiphyseal dysplasia (MED), which is characterized by short stature and early-onset osteoarthritis, and bilateral hereditary microepiphyseal dysplasia, a variant form of MED characterized by pain in the hip and knee joints. To assess the function of matrilin-3 during skeletal development, we have generated Matn-3 null mice. Homozygous mutant mice appear normal, are fertile, and show no obvious skeletal malformations. Histological and ultrastructural analyses reveal endochondral bone formation indistinguishable from that of wild-type animals. Northern blot, immunohistochemical, and biochemical analyses indicated no compensatory upregulation of any other member of the matrilin family. Altogether, our findings suggest functional redundancy among matrilins and demonstrate that the phenotypes of MED disorders are not caused by the absence of matrilin-3 in cartilage ECM.     

Retinal and optical adaptations for nocturnal vision in the halictid bee<Emphasis Type="Italic"> Megalopta genalis</Emphasis>

The apposition compound eye of a nocturnal bee, the halictid Megalopta genalis, is described for the first time. Compared to the compound eye of the worker honeybee Apis mellifera and the diurnal halictid bee Lasioglossum leucozonium, the eye of M. genalis shows specific retinal and optical adaptations for vision in dim light. The major anatomical adaptations within the eye of the nocturnal bee are (1) nearly twofold larger ommatidial facets and (2) a 4–5 times wider rhabdom diameter than found in the diurnal bees studied. Optically, the apposition eye of M. genalis is 27 times more sensitive to light than the eyes of the diurnal bees. This increased optical sensitivity represents a clear optical adaptation to low light intensities. Although this unique nocturnal apposition eye has a greatly improved ability to catch light, a 27-fold increase in sensitivity alone cannot account for nocturnal vision at light intensities that are 8 log units dimmer than during daytime. New evidence suggests that additional neuronal spatial summation within the first optic ganglion, the lamina, is involved.B.G. is thankful for travel awards from the Royal Physiographic Society, the Per Westlings Fond, the Foundation of Dagny and Eilert Ekvall and the Royal Swedish Academy of Sciences. E.J.W. is grateful for the support of a Smithsonian Short-Term Research Fellowship, the Swedish Research Council, the Crafoord Foundation, the Wenner-Gren Foundation and the Royal Physiographic Society of Lund for their ongoing support     

Structure and function of vertebrate CMP-sialic acid synthetases

Activation of sugars into nucleotide sugars is critical for their entry into biosynthetic pathways. In eukaryotic cells, the activation of the acidic nine-carbon sugar sialic acid to CMP-sialic acid takes place in the cell nucleus, whereas all other nucleotide sugars are made in the cytoplasm. Molecular cloning of vertebrate CMP-sialic acid synthetases confirmed the nuclear localization and introduced new molecular tools for directly exploring the functional mechanisms of the enzymes, as well as the physiological relevance of their nuclear transport. Although major advances have been made in understanding structure-function relationships and defining elements involved in the nuclear transport, the riddle surrounding the physiological relevance of nuclear localization awaits resolution.     

Stable haploid poplar callus lines from immature pollen culture

Embryogenesis and plant regeneration have been obtained from isolated immature pollen of two poplar hybrids ( Populus nigra L. × hybrid 'Aue1' and 'Aue2'). In total, 1487 calli or embryos, respectively, larger than 1 mm were generated in a 2-year study. By using a cytokinin containing induction medium, on average 19 calli per responsive immature catkin were formed. Additional supplementation with auxin in 2002 increased the frequency to 72 calli per catkin. Microsatellite marker analyses confirmed haploid origin in most regenerants studied. So far six out of eight obtained regenerative callus lines have maintained their haploid level up to 24 months of development. A number of haploid and doubled haploid plants of different lines are available and have been transferred to soil.     

The bacterial paromomycin resistance gene, aphH, as a dominant selectable marker in Volvox carteri

The aminoglycoside antibiotic paromomycin that is highly toxic to the green alga Volvox carteri is efficiently inactivated by aminoglycoside 3′-phosphotransferase from Streptomyces rimosus. Therefore, we made constructs in which the bacterial aphH gene encoding this enzyme was combined with Volvox cis-regulatory elements in an attempt to develop a new dominant selectable marker – paromomycin resistance (Pm^R) – for use in Volvox nuclear transformation. The construct that provided the most efficient transformation was one in which aphH was placed between a chimeric promoter that was generated by fusing the Volvox hsp70 and rbcS3 promoters and the 3′ UTR of the Volvox rbcS3 gene. When this plasmid was used in combination with a high-impact biolistic device, the frequency of stable Pm^R transformants ranged about 15 per 10⁶ target cells. Due to rapid and sharp selection, Pm^R transformants were readily isolated after six days, which is half the time required for previously used markers. Co-transformation of an unselected marker ranged about 30%. The chimeric aphH gene was stably integrated into the Volvox genome, frequently as tandem multiple copies, and was expressed at a level that made selection of Pm^R transformants simple and unambiguous. This makes the engineered bacterial aphH gene an efficient dominant selection marker for the transformation and co-transformation of a broad range of V. carteri strains without the recurring need for using auxotrophic recipient strains.     

The complete genome sequence of Bacillus licheniformis DSM13, an organism with great industrial potential

The genome of Bacillus licheniformis DSM13 consists of a single chromosome that has a size of 4,222,748 base pairs. The average G+C ratio is 46.2%. 4,286 open reading frames, 72 tRNA genes, 7 rRNA operons and 20 transposase genes were identified. The genome shows a marked co-linearity with Bacillus subtilis but contains defined inserted regions that can be identified at the sequence as well as at the functional level. B. licheniformis DSM13 has a well-conserved secretory system, no polyketide biosynthesis, but is able to form the lipopeptide lichenysin. From the further analysis of the genome sequence, we identified conserved regulatory DNA motives, the occurrence of the glyoxylate bypass and the presence of anaerobic ribonucleotide reductase explaining that B. licheniformis is able to grow on acetate and 2,3-butanediol as well as anaerobically on glucose. Many new genes of potential interest for biotechnological applications were found in B. licheniformis; candidates include proteases, pectate lyases, lipases and various polysaccharide degrading enzymes.