Structural analysis of Arabidopsis thaliana chromosome 5. X. Sequence features of the regions of 3,076,755 bp covered by sixty P1 and TAC clones.

In our ongoing project to deduce the nucleotide sequence of Arabidopsis thaliana chromosome 5, non-redundant P1 and TAC clones have been sequenced on the basis of the fine physical map, and as of January, 2000, the sequences of 16.6 Mb representing approximately 60% of chromosome 5 have been accumulated and released at our web site. Along with the sequence determination, structural features of the sequenced regions have been analyzed by applying a variety of computer programs, and we already predicted a total of 2697 potential protein coding genes in the 11,166,130 bp regions, which are covered by 159 P1 and TAC clones. In this paper, we describe the structural features of the 3,076,755 bp regions covered by newly analyzed 60 P1 and TAC clones. A total of 715 potential protein coding genes were identified, giving an average density of the genes identified of 1 gene per 4001 bp. Introns were observed in 80% of the genes, and the average number per gene and the average length of the introns were 4.5 and 147 bp, respectively. These sequence features are nearly identical to those in our latest report in which the data were compiled based on a new standard of gene assignment including the computer-predicted hypothetical genes. The regions also contained 12 tRNA genes when searched by similarity to reported tRNA genes and the tRNA scan-SE program. The sequence data and information on the potential genes are available through the World Wide Web database KAOS (Kazusa Arabidopsis data Opening Site) at http://www.kazusa.or.jp/kaos/.     

Protein Tyrosine Kinase cDNAs from Amphioxus, Hagfish, and Lamprey: Isoform Duplications Around the Divergence of Cyclostomes and Gnathostomes

Animals evolved a variety of gene families involved in cell–cell communication and developmental control by gene duplication and domain shuffling. Each family is made up of several subtypes or subfamilies with distinct structures and functions, which diverged by gene duplications and domain shufflings before the divergence of parazoans and eumetazoans. Since the separation from protostomes, vertebrates expanded the multiplicity of members (isoforms) in the same subfamily by further gene duplications in their early evolution before the fish–tetrapod split. To know the dates of isoform duplications more closely, we have conducted isolation and sequencing cDNAs encoding the fibroblast growth factor receptor, Eph, src, and platelet-derived growth factor receptor subtypes belonging to the protein tyrosine kinase family from Branchiostoma belcheri, an amphioxus, Eptatretus burgeri, a hagfish, and Lampetra reissneri, a lamprey. From a phylogenetic tree of each subfamily inferred from a maximum likelihood (ML) method, together with a bootstrap analysis based on the ML method, we have shown that the isoform duplications frequently occurred in the early evolution of vertebrates around or just before the divergence of cyclostomes and gnathostomes by gene duplications and possibly chromosomal duplications. Received: 28 April 1998 / Accepted: 30 June 1999     

Alleic variants of human melatonin 1a receptor: function and prevalence in subjects with circadian rhythm sleep disorders.

The human melatonin 1a (hMella) receptor gene was screened for mutations using genomic DNA samples from patients with circadian rhythm sleep disorders and control subjects by single strand conformational polymorphism analysis (SSCP). We found seven mutations, two of which predict amino acid changes R54W and A157V, respectively. The prevalence of the R54W variant and that of the A157V variant were several times more common in non-24-h sleep-wake syndrome subjects than among control subjects, although the incidence was not significant in our study group. When expressed in COS-7 cells, the R54W mutant receptor exhibited significantly reduced B(max) and slightly enhanced affinity (reduced K(d)) compared to the wild type receptor, while the A157V variant receptor showed similar binding characteristics to the wild type. The identification of variants in the hMella receptor will provide a useful tool for analyzing genetic predisposition toward various diseases related to melatonin function and to clarify the physiological role of melatonin receptors in humans.     

Biological activities of binding site specific monoclonal antibodies to prolactin receptors of rabbit mammary gland

The biological activity of three monoclonal antibodies (mAbs) against the rabbit mammary prolactin (PRL) receptor (M110, A82, and A917) were investigated using explants of rabbit mammary gland. The three mAbs which were all able to inhibit the binding of 125I-ovine prolactin to its receptor had different biological activities. Two mAbs (M110 and A82) were able to prevent the stimulating effect of PRL on casein synthesis when the molar ratio between the mAb and PRL was 100. At a lower concentration, M110 moved the PRL dose-response curve to the right by a factor of 2.4. This mAb was also effective in vivo, reducing milk production in a lactating rabbit, in a similar fashion to the prolactin lowering drug, CB-154. One mAb (A917) was able to mimic the action of PRL on both casein and DNA ([3H]thymidine incorporation) synthesis, whereas the other two mAbs were without any stimulatory effect. For this stimulatory effect to be observed, bivalency of the antibody was essential, since monovalent fragments, which were able to inhibit PRL binding, had no agonistic activity. The ability of the mAbs to induce a down-regulation of receptors was also studied. M110, which was equipotent to PRL in occupation of receptors, induced no down-regulation, while A917, which had full biological activity, induced only a small degree of down-regulation. These studies suggest that the binding domain of the receptor might be relatively complex, since only a part of this domain recognized by the antibody with PRL-like activity was able to induce hormonal action. Alternatively, only those antibodies able to microaggregate the receptors may possess PRL-like activity.     

Topographical Studies on Subunit Polypeptides of the PS I Reaction Center Complex in the Thylakoid Membranes of the Thermophilic Cyanobacterium Synechococcus sp.

The permeability to protein molecules of the outer limitingmembranes and the thylakoid membranes in hypotonically shockedprotoplasts of the thermophilic cyanobacterium Synechococcussp. was studied by examining the effects of NaBr-washing andpronase E-digestion on phycobiliproteins and a 35 kDa proteinwhich are associated with the outer and inner surface of thethylakoid membranes, respectively, and by measuring photooxidationof added cytochrome c. All the results obtained indicate thatthe shocked protoplasts are in essence a homogenous right side-outthylakoid membrane preparation; the outer limiting membranesare leaky to protein molecules, whereas the thylakoid membranesare still impermeable to proteins. The thylakoid membranes becamepermeable to proteins when the protoplasts were mechanicallydisrupted. Following on from these findings, the membrane topology of subunitpolypeptides of the photosystem I reaction center complex wasstudied. Proteolytic digestion of shocked protoplasts with trypsinand pronase E indicated that four of the five subunit polypeptidesof the PS I reaction center complex are exposed at the stromalsurface of thylakoid membranes; two subunits of 14 and 13 kDawere selectively digested by trypsin, whereas two chlorophyll-bindingsubunits of 62 and 60 kDa were preferentially attacked by pronaseE. However, a 10 kDa subunit appears to be strongly resistantto the proteases. Experiments with mechanically disrupted protoplastsfailed to provide evidence for a uniform transmembrane organizationof the PS I subunits. (Received March 31, 1986; Accepted August 18, 1986)     

Characterization of the growth responses of hamster fibroblasts and chondrogenic cells to fibroblast growth factor

The effects of fibroblast growth factor (FGF) on hamster dermal fibroblasts and chondrogenic cells, both of mesodermal origin, were compared with special reference to growth stimulation and morphological changes in monolayer cultures, and colony formation in semisolid medium. FGF (10 to 200 ng/ml) caused appreciable cell proliferation of dermal fibroblasts but not of chondrogenic cells, while FGF (50-200 ng/ml) caused very marked dose-dependent morphological changes in monolayer cultures and colony formation in semisolid medium of both fibroblasts and chondrogenic cells. It is suggested that FGF is the same type of growth factor as the transforming growth factor(s) because, like the latter, it induces drastic morphological changes of normal mesodermal cells in monolayer cultures and their colony formation in semisolid medium.     

Chlorophyll b-Deficient Mutants of Rice: I. Absorption and Fluorescence Spectra and Chlorophyll a/b Ratios

Absorption spectra and their fourth derivatives of chloroplastsisolated from 16 different rice chlorina mutants were determinedat liquid nitrogen temperature. The results suggest that Chlb is absent from 10 mutants labelled chlorina-1 to -10, while6 mutants named chlorina-11 to -16 contain low levels of Chlb. Low temperature fluorescence emission spectra indicate thata light-harvesting Chl a/b protein of photosystem I is presentin chlorina-11 to -16 but not in chlorina-1 to -10. Reinvestigationof Chl a/b ratios by a sensitive fluorescence method shows thatthe 16 mutants are divided into three groups different in thedegree of Chl b-deficiency; chlorina-1 to -10 totally lack Chlb (Type I), chlorina-11 to -13 have Chl a/b ratio of about 10(Type II-A) and chlorina-14 to -16 have the ratio of about 15(Type II-B). (Received June 6, 1985; Accepted August 6, 1985)     

Chlorophyll b-Deficient Mutants of Rice: II. Antenna Chlorophyll a/b-Proteins of Photosystem I and II

Chlorophyll-protein complexes of the wild type and 16 strainsof chlorina mutants of rice were investigated by gel electrophoresis.An antenna chlorophyll a/b-protein of photosystem II (LHC-II)was present in reduced amounts in Type II chlorina mutants whichhave the chlorophyll a/b ratios of 10–15, and was totallyabsent from Type I chlorina mutants which lack chlorophyll b.Another antenna chlorophyll-protein of photosystem I (LHC-I)containing two polypeptides of 20 and 21 kDa was also presentin the Type II mutants but not in the Type I mutants. The polypeptideprofiles of the thylakoid membranes indicate that Type I mutantslack both the 20 and 21 kDa polypeptides, whereas the abundanceof the two polypeptides relative to the CPI apoprotein in theType II mutants is comparable with that in the wild type. Itis concluded that the 20 and 21 kDa polypeptides are both relatedto LHC-I and are normally synthesized and accumulated in theType II mutants. (Received June 6, 1985; Accepted August 6, 1985)     

Reaction kinetics of P-700, cytochrome c-553 and cytochrome f in the cyanobacterium, Synechococcus sp

Absorption changes invoked by short flashes in the Soret band region were measured in the thermophilic cyanobacterium Synechococcus sp. and photoresponses of P-700, cytochrome c-553 and cytochrome f were resolved with the aid of a microcomputer. Cytochrome c-553 was oxidized very rapidly with a half-time of less than 20 μs, while the half oxidation time of cytochrome f was 35–45 μs. The two cytochromes were reduced monophasically with half-time of 2 ms after a lag lasting a few milliseconds. The reduction kinetics of P-700 showed three exponential phases with half-times of 40 μs, 200 μs and 2 ms, which are ascribed to electron donation from cytochrome f, the Rieske iron-sulfur protein and plastoquinone, respectively. The results support the following sequence and rates of linear electron transport at the physiological temperature of the cyanobacterium: P-700

cytochrome c-553

cytochrome f

Rieske protein

plastoquinone.     

Phase-linked variations in the amplitude of the digastric nerve jaw-opening reflex response during fictive mastication in the rabbit

The diagastric nerve reflex response to stimulation of the upper lip was studied in urethan-anesthetized rabbits paralysed with pancuronium bromide. Rhythmic bursts of masticatory activity were evoked in the nerve by repetitive electrical stimulation of the motor cortex. The amplitude and latency of the reflex responses during fictive mastication were compared with preceding control values. When stimuli close to threshold were given, the largest and earliest responses occurred during the digastric burst. When intense stimuli were employed, the largest responses were out of phase with the burst, although the latency was still shortest when the motoneurons were rhythmically active. Since the pattern is essentially the same as that seen during normal mastication, we conclude that the cyclical modulation of reflex amplitude and latency is not the result of sensory feedback generated by the movements themselves but is instead governed by the central motor program.