Regulation of influenza virus RNA polymerase activity by cellular and viral factors.

An in vitro RNA synthesis system mimicking replication of genomic influenza virus RNA was developed with nuclear extracts prepared from influenza virus-infected HeLa cells using exogenously added RNA templates. The RNA synthesizing activity was divided into two complementing fractions, i.e. the ribonucleoprotein (RNP) complexes and the fraction free of RNP, which could be replaced with RNP cores isolated from virions and nuclear extracts from uninfected cells, respectively. When nuclear extracts from uninfected cells were fractionated by phosphocellulose column chromatography, the stimulatory activity for RNA synthesis was further separated into two distinct fractions. One of them, tentatively designated RAF (RNA polymerase activating factor), stimulated RNA synthesis with either RNP cores or RNA polymerase and nucleocapsid protein purified from RNP cores as the enzyme source. In contrast, the other, designated PRF (polymerase regulating factor), functioned as an activator only when RNP cores were used as the enzyme source. Biochemical analyses revealed that PRF facilitates dissociation of RNA polymerase from RNP cores. Of interest is that virus-coded non-structural protein 1 (NS1), which has been thought to be involved in regulation of replication, counteracted PRF function. Roles of cellular factors and viral proteins, NS1 in particular, are discussed in terms of regulation of influenza virus RNA genome replication.     

Solution structure of the epidermal growth factor-like domain of heregulin-alpha, a ligand for p180erbB-4.

p185erbB-2 and p180erbB-4 are epidermal growth factor (EGF) receptor-like tyrosine kinases, whose co-expression is observed in many breast carcinomas. Heregulins (HRGs), which contain an immunoglobulin unit and an EGF-like domain, bind to p180erbB-4 and activate p180erbB-4 and p185erbB-2 through transphosphorylation or receptor heterodimerization. The EGF-like domain is sufficient for the activation. Despite the sequence similarity, no cross activity is seen between the p180erbB-4 ligands (HRGs) and the p170erbB-1 ligands [EGF and transforming growth factor (TGF)-alpha]. To investigate the structural basis of receptor specificity, we have determined the solution structure of the EGF-like domain of HRG-alpha by two-dimensional 1H nuclear magnetic resonance spectroscopy and simulated annealing calculations. Though its main-chain fold is similar to those of EGF and TGF-alpha, distinctive structural features are observed on the molecular surface including an ionic cluster and hydrophobic patches, which afford HRG-alpha the specific affinity for p180erbB-4. The structure should provide a basis for the structure-activity relationship of HRGs and for the design of drugs which prevent progression of breast cancer.     

Alternative 5'' splice site selection induced by heat shock.

The mouse HSP47 gene consists of six exons separated by five introns. Three HSP47 cDNAs differing only in their 5' noncoding regions have been reported. One of these alternatively spliced mRNAs was detected only after heat shock, which caused an alternative 5' splice donor site selection. Other stress inducers, including an amino acid analog and sodium arsenite, had no effect on the alternative splicing. The alternatively spliced mRNA, which was 169 nucleotides longer in the 5' noncoding region compared to mRNA transcribed in non-heat shock conditions, was efficiently translated under heat shock conditions. This novel finding that alternative splicing is caused by artificial treatment like heat shock will provide a useful in vivo model for understanding the exon-intron recognition mechanism as well as heat shock-induced alterations in gene expression.     

Nucleotide sequence and functional analysis of the meta-cleavage pathway involved in biphenyl and polychlorinated biphenyl degradation in Pseudomonas sp. strain KKS102.

Pseudomonas sp. strain KKS102 is able to degrade biphenyl and polychlorinated biphenyls via the meta-cleavage pathway. We sequenced the upstream region of the bphA1A2A3BCD (open reading frame 1 [ORF1]) A4 and found four ORFs in this region. As the deduced amino acid sequences of the first, second, and third ORFs are homologous to the meta-cleavage enzymes from Pseudomonas sp. strain CF600 (V. Shingler, J. Powlowski, and U. Marklund, J. Bacteriol. 174:711-724, 1992), these ORFs have been named bphE, bphG, and bphF, respectively. The fourth ORF (ORF4) showed homology with ORF3 from Pseudomonas pseudoalcaligenes KF707 (K. Taira, J. Hirose, S. Hayashida, and K. Furukawa, J. Biol. Chem. 267:4844-4853, 1992), whose function is unknown. The functions of meta-cleavage enzymes (BphE, BphG, and BphF) were analyzed by using crude extracts of Escherichia coli which expressed the encoding genes. The results showed that bphE, bphG, and bphF encode 2-hydroxypenta-2,4-dienoate hydratase, acetaldehyde dehydrogenase (acylating), and 4-hydroxy-2-oxovalerate aldolase, respectively. The biphenyl and polychlorinated biphenyl degradation pathway of KKS102 is encoded by 12 genes in the order bphEGF (ORF4)A1A2A3BCD (ORF1)A4. The functions of ORF1 and ORF4 are unknown. The features of this bph gene cluster are discussed.     

Dispersal of wild and domestic masu salmon fry (Oncorhynchus masou) in an artificial channel

The day and night pattern of upstream and downstream dispersal of masu salmon fry of wild and domestic origin was compared in artificial channels (45 m long), for two ages of planting: unfed alevins and eyed eggs. Early dispersal was important for the wild stock (48–50%) compared with the domestic one (16–36%). More wild fry moved downstream than upstream, and more domestic fry dispersed upstream. Upstream movement in wild and domestic fry was more active by day than by night, except for wild fry planted as eyed eggs, where upstream migration was higher at night. In contrast, downstream movement in wild and domestic fry was more common by night than by day, but daylight catches were not negligible for the wild stock.     

Intraspecific differences in patterns of courtship behaviours between the Pacific Ocean and Japan Sea forms of the three-spined stickleback Gasterosteus aculeatus

The courtship behaviour of the Pacific Ocean and Japan Sea forms of the three-spined stickleback Gasterosteus aculeatus were studied in seven populations. The intraspecific difference in behavioural patterns, zigzag dance and weak dorsal pricking in the Pacific Ocean form and lateral display and vigorous dorsal pricking in the Japan Sea form, was clearly distinguishable regardless of the type of habitat the males lived in.     

Cell wall regeneration and cell division in isolated tobacco mesophyll protoplasts

Summary Protoplasts were isolated from palisade tissue of tobacco leaves by treatment with pectinase and cellulase under aseptic conditions, and were cultured in a synthetic liquid medium. Calcofluor, a fluorescent brightener, was found to be an excellent stain for plant cell walls and was used to demonstrate regeneration of cell walls in these protoplasts. The cultured protoplasts regenerated cell walls by the 3rd day of culture, giving rise to spherical cells. The majority of the protoplasts regenerating cell walls underwent mitosis and cell division. The cycle of mitosis and cell division was repeated 2–3 times during 2 weeks of culture. Some of the nutritional conditions affecting division in the cultured protoplasts were studied.