Cloning of the Gene Encoding a Protochlorophyllide Reductase: the Physiological Significance of the Co-Existence of Light-Dependent and -Independent Protochlorophyllide Reduction Systems in the Cyanobacterium Plectonema boryanum

Cyanobacteria have two protochlorophyllide (Pchlide) reductasescatalyzing the conversion of Pchlide to chloro-phyllide, a keystep in the biosynthetic pathway of chlorophylls (Chls); a light-dependent(LPOR) and a light-independent (DPOR) reductase. We found anopen reading frame (ORF322) in a 2,131-bp EcoRI fragment fromthe genomic DNA of the cyanobacterium Plectonema boryanum. Becausethe deduced amino acid sequence showed a high similarity tothose of various plant LPORs and the LPOR activity was detectedin the soluble fraction of Esche-richia coli cells over-expressingthe ORF322 protein, ORF322 was defined as the por gene encodingLPOR in P. boryanum. A por-disrupted mutant, YFP12, was isolatedby targeted mutagenesiss to investigate the physiological importanceof LPOR. YFP12 grew as well as wild type under low light conditions(10-25 µE m^–2 S^–1). However, its growth wassignificantly retarded as a result of a significant decreasein its Chl content under higher light conditions (85-130 µEm^–2 s^–1). Furthermore, YFP12 stopped growing andsuffered from photobleaching under the highest light intensity(170 µE m^–2 s^–1). In contrast, a chlL-dis-rupted(DPOR-less) mutant YFC2 grew as well as wild type irrespectiveof light intensity. From these phenotypic characteristics, weconcluded that, although both LPOR and DPOR contribute to Chlsynthesis in the cells growing in the light, the extent of thecontribution by LPOR increases with increasing light intensity;without it, the cells are unable to grow under light intensitiesof more than 130 µ Em^–2s-^–. (Received September 26, 1997; Accepted November 21, 1997)     

Origin and divergence of tandem repeats of primate D4 dopamine receptor genes

Human D4 dopamine receptor (D4DR) is polymorphic in terms of the repeat, numbers of the 48-base pairs (bp) sequence located in the third cytoplasmic loop of the receptor. The repeated sequence and its polymorphism in D4DR genes have also been identified in higher non-human primates, suggesting that the structure of D4DR has been maintained during primate evolution. To clarify the origin and divergence of the polymorphism in the D4DR gene, we determined the nucleotide sequence of this region of the D4DR gene in several species of prosimians and the tree shrew, a species which is closely related to primates. Prosimians except the tarsier had one or two unit(s) of the 48-bp sequence, and conserved sequences were recognized in most of the units of the prosimians. The tree shrew had only one unit of the 48-bp sequence, and its sequence was 71–75% identical to those of the nuits of galago, loris, and lemur. These findings suggest that the ancestral primate presumably had one 48-bp unit, and duplication of the unit occurred at the stage of prosimians. Tarsiers appeared to be distinct from other prosimians and simians because of the high repeat numbers of units and their sequences.     

The Promoter of a Pine Photosynthetic Gene Allows Expression of a {beta}-Glucuronidase Reporter Gene in Transgenic Rice Plants in a Light-Independent but Tissue-Specific Manner

In angiosperms, the expression of the cab gene that encodesthe chlorophyll a/b-binding protein of PSII is light-regulated.However, the pine cab gene is expressed in a light-independentbut cell-type-specific manner. In the present study, the cab-6promoter (1.7 kbp) from pine was fused to a -glucuronidase (GUS)reporter gene and the chimeric gene was introduced into riceprotoplasts by electroporation. The GUS expression was studiedin the resultant transgenic rice plants. Expression of GUS ata substantial level was confirmed in primary leaves of dark-germinatedrice seedlings, and no obvious effect of light on the GUS activitywas observed. The expression of GUS was restricted to photosynthetictissues. The pine cab-6 promoter is, thus, sufficient for inductionof light-independent but cell-type-specific expression in cellsof a monocot, as is the case in the original pine cells. (Received December 17, 1993; Accepted April 22, 1994)     

A novel biotinylated heterobifunctional cross-linking reagent bearing an aromatic diazirine

The synthesis of a p-[(3-trifluoromethyl)diazirine-3-yl]benzoic acid derivative is described as a new carbene generating heterobifunctional cross-linking reagent. The cross-linker carries a biotin moiety in order to make use of avidin—biotin technology for specific manipulation of cross-linked components. To evaluate the ability of this reagent, the inter-subunit cross-linking of egg-white avidin tetramer was investigated. As a typical application of avidin—biotin technology for cross-linking experiments, a chemiluminescent detection method was examined to identify photobiotinylated components. A cross-linked dimeric product with an apparent molecular mass of 38 kDa was clearly visualized by the combined use of a horseradish peroxidase—streptavidin conjugate and a luminol-based chemiluminescent system.     

C55 bacteriocin produced by ETB‐plasmid positive Staphylococcus aureus strains is a key factor for competition with S. aureus strains

Exfoliative toxin (ET) produced by Staphylococcus aureus is closely associated with the onset of bullous impetigo. To date, three ETs (ETA, ETB and ETD) have been identified. The gene encoding ETB is located in a plasmid designated pETB. Bacteriocin synthesis genes are also located in this plasmid and pETB‐positive strains reportedly produce the C55 bacteriocin. In this study, the antibacterial activity against S. aureus strains of the bacteriocin produced by the pETB‐positive strain TY4 was investigated. This bacteriocin demonstrated antibacterial activity against all pETB‐negative but not pETB‐positive strains, including TY4. Additionally, a TY4? strain from which the pETB plasmid had been deleted exhibited susceptibility to the bacteriocin. Further experiments revealed that two immunity factors (orf 46‐47 and orf 48) downstream of the bacteriocin synthesis genes in the pETB plasmid are associated with immunity against the bacteriocin produced by TY4. The TY4? with orf46‐47 strain exhibited complete resistance to bacteriocin, whereas the TY4? with orf48 strain exhibited partial resistance. Whether bacteriocin affects the proportion of each strain when co‐cultured with S. aureus strains was also investigated. When TY4 or TY4? was co‐cultured with 209P strain, which is susceptible to the bacteriocin, the proportion of 209P co‐cultured with TY4 was significantly less than when 209P was co‐cultured with TY4?, whereas the proportion of TY4? with orf46‐48 co‐cultured with TY4 was greater than with TY4?. These results suggest that the C55 bacteriocin produced by pETB‐positive strains affects the proportion of each strain when pETB‐positive and ‐negative strains co‐exist.