Characterization and mapping of Ds—GUS-T-DNA lines for targeted insertional mutagenesis

The transposition patterns of the Ds —GUS transposon T-DNA in 23 independent single-copy lines have been characterized and the map positions of 10 of them on three of the five Arabidopsis chromosomes are reported. Using overexpressed Activator ( Ac ) elements as a transposase source, it was found that the primary determinant of transposition frequency is the insertion site of the Ac -T-DNA. Neither the structure of the transposon T-DNA nor, in most cases, its insertion site have a significant effect on transposition frequency. Both the frequency and timing of transposition are influenced by the parent through which the transposon and transposase T-DNAs are transmitted. Overall, nearly 75% of plants in which excision has occurred bear a reinserted element and very short-range transpositions predominate, underlining the advantage of using mapped transposons for insertional mutagenesis.     

Water Soluble Pigments Containing Xylose and Glucose in Gametangia of the Green Alga, Bryopsis maxima

Several water-soluble pigments were purified from gametangiaof Bryopsis maxima by liquid chromatography and characterizedby pyridylamination and high-performance anion-exchange chromatography.The structure of the main red pigment is proposed based on thedata of infrared spectrum, Mass spectrum, ¹H and ¹³C NMR spectraand pyridylamino analysis. As a consequence, this pigment containeda tetrapyrrole with phytol and a sugar chain comprised of xyloseand glucose. The sequence of the sugars in the chain was determinedbased on its Mass spectrum. The pigment was similar to chlorophyll-originpigments observed in other plants. No aldehyde group, however,was present at C5 in the open tetrapyrrole chain. (Received August 3, 1994; Accepted November 10, 1994)     

Two-step regulation of ecdysone-inducible late puffs in salivary glands of Drosophila melanogaster

Our previous study showed that some ecdysone-inducible late puffs could also be induced by a mild detergent (digitonin) in Drosophila salivary glands. However, they could only be induced at the stage immediately prior to when developmentally programmed puffing occurred, suggesting that these late puff loci were under two-step regulation. Using an in vitro culture of salivary glands, we have examined whether ecdysone or the protein products of early puff genes participate in either of the two steps of late puff regulation. This study has revealed that (i) the acquisition of digitonin-responsiveness (the first step) could be induced in vitro by incubating salivary glands with ecdysone; (ii) the first step could also be induced by protein synthesis inhibition even in the absence of ecdysone; (iii) the second step required both ecdysone and protein synthesis unless treated with digitonin; and (iv) the first step, rather than the second step, determines the timing of normal puff formation in the loci. These results suggest that, during normal development, ecdysone controls both steps by activating two types of early genes; the first type, whose function can be mimicked by cycloheximide, renders the loci responsive to digitonin and the second type, whose function can be mimicked by digitonin, activates the loci to form puffs.     

Characterization of a gene cluster for glycogen biosynthesis and a heterotetrameric ADP-glucose pyrophosphorylase from Bacillus stearothermophilus.

A chromosomal region of Bacillus stearothermophilus TRBE14 which contains genes for glycogen synthesis was cloned and sequenced. This region includes five open reading frames (glgBCDAP). It has already been demonstrated that glgB encodes branching enzyme (EC 2.4.1.18 [H. Takata et al., Appl. Environ. Microbiol. 60:3096-3104, 1994]). The putative GlgC (387 amino acids [aa]) and GlgD (343 aa) proteins are homologous to bacterial ADP-glucose pyrophosphorylase (AGP [EC 2.7.7.27]): the sequences share 42 to 70% and 20 to 30% identities with AGP, respectively. Purification of GlgC and GlgD indicated that AGP is an alpha2beta2-type heterotetrameric enzyme consisting of these two proteins. AGP did not seem to be an allosteric enzyme, although the activities of most bacterial AGPs are known to be allosterically controlled. GlgC protein had AGP activity without GlgD protein, but its activity was lower than that of the heterotetrameric enzyme. The GlgA (485 aa) and GlgP (798 aa) proteins were shown to be glycogen synthase (EC 2.4.1.21) and glycogen phosphorylase (EC 2.4.1.1), respectively. We constructed plasmids harboring these five genes (glgBCDAP) and assayed glycogen production by a strain carrying each of the derivative plasmids on which the genes were mutated one by one. Glycogen metabolism in B. stearothermophilus is discussed on the basis of these results.     

Computer determination of perfusion patterns in pulmonary capillary networks

Hanger, Christopher C., Robert G. Presson, Jr., Osamu Okada,Steven J. Janke, John J. Watkins, Wiltz W. Wagner, Jr., and Ronald L. Capen. Computer determination of perfusion patterns in pulmonarycapillary networks. J. Appl. Physiol.82(4): 1283-1289, 1997.Individual pulmonary capillaries are notsteadily perfused. By using in vivo microscopy, it can readily bedemonstrated that perfusion continually switches between capillarysegments and between portions of the network within a single alveolarwall. These changes in capillary perfusion occur even when upstream pressure and flow are constant. Flow switching between capillary segments in the absence of hemodynamic changes in large upstream vessels suggests that capillary perfusion patterns could be random. Tocalculate the probability that perfusion patterns could occur bychance, it is necessary to know the total number of possible perfusionpatterns in a given capillary network. We developed a computer programthat can determine every possible perfusion pattern for any givencapillary network, and from that information we can calculate whetherperfusion of individual segments in the network is random. With theresults of the computer program, we have obtained statistical evidencethat some capillary segments in a network are nonrandomly perfused.

     

Hydrocarbon composition of newly isolated strains of the green microalga Botryococcus braunii

Four new strains of Botryococcus braunii were isolated from Japanese waters and cultured under defined conditions. Their hydrocarbon content and composition were analyzed and compared with those of the Darwin and Berkeley strains. The Yamanaka strain produced only alkadienes characteristic of the A race, whereas the others, the Yayoi, Kawaguchi-1 and -2 strains as well as the Darwin and Berkeley strains, produced botryococcenes peculiar to the B race. The hydrocarbon content of the Yamanaka strain was 16.1 % dry weight and that of the B race strains ranged from 9.7 to 37.9%. Botryococcene composition of the Japanese strains differed from each other as well as from the Darwin and Berkeley strains. More than 50% of the hydrocarbons in the Yayoi, Darwin, and Berkeley strains were composed of C₃₄H₅₈, but the main components were different from one another as isomers. The Kawaguchi-1 and -2 strains did not have a high level of C₃₄ botryococcenes, C₃₂ ones being the main components. In these strains significant amounts of squalene-related compounds were detected.     

Role of the conserved C-repeat region of the M protein of Streptococcus pyogenes

The surface-located M protein functions to protect Streptococcus pyogenes (the group A streptococcus) from phagocytosis by polymorphonuclear leukocytes. It has been suggested that this protection results from the ability of M protein to bind factor H, a serum protein that can inhibit the activation of complement. Among different serological variants of M protein, the C-repeat domain is highly conserved and is exposed on the bacterial surface. This domain has been implicated in binding to complement factor H and in M-protein-mediated adherence of streptococci to human keratinocytes in the cutaneous epithelium. In this study, we constructed an S. pyogenes mutant strain which expresses an M6 protein from which the entire C-repeat domain was deleted. As predicted, this mutant did not adhere well to human keratinocytes and was unable to bind to factor H. Unexpectedly, the mutant was able to survive and multiply in human blood. Therefore, while the binding of factor H and the facilitation of adherence to keratinocytes appear to involve recognition of the C-repeat domain, a region of the M-protein molecule distinct from the C-repeat domain confers upon S. pyogenes its ability to resist phagocytosis.     

Differentiation-dependent expression of the chicken delta-crystallin gene introduced into mouse teratocarcinoma stem cells.

PCC3 mouse teratocarcinoma (TCC) stem cells were cotransfected with either the plasmid p delta C-1A or p delta C-1B carrying the chicken delta-crystallin gene, and with the plasmid pSV2gpt containing the selectable bacterial xanthine-guanine phosphoribosyltransferase (XGPRT) gene, using the calcium phosphate technique. Nine transformed PCC3 stem cell lines, each of which was clonally derived from respective colonies surviving after the selection process, were isolated. Southern blot analysis revealed that all of them stably maintained delta-crystallin sequences associated with high mol. wt. cellular DNA after propagation in non-selective medium in vitro, and after the production of solid tumors in the syngenic host mice. Six cell lines contain the intact delta-crystallin gene sequence and eight contain the gpt sequence. The number of delta-crystallin DNA copies was highly variable among transformed lines, 1-500 delta-crystallin genes per diploid mouse genome. No expression of the exogenous genes was detected in the transformed cells as long as they were in the undifferentiated state. However, the synthesis of delta-crystallin in certain types of cells was detected immunohistologically in three lines after the differentiation. The positive cell types were unique to each line, skeletal muscle in Y delta-9, certain columnar epithelia in Y delta-2, and unidentified spindle-shaped cells in Y delta-3. Authentic delta-crystallin polypeptides with a mol. wt. of 48 000 are synthesized upon differentiation of line Y delta-3 in solid tumors in syngenic mice.     

The nucleotide sequence of a complete chicken delta-crystallin cDNA

The nucleotide sequence of a full length cDNA of delta-crystallin mRNA from chicken lens has been determined using a delta-crystallin cDNA clone (pB delta 11), which represents the mRNA sequence of 1530 nucleotides from the poly(A) junction but does not contain the 5'-terminal sequence of 44 nucleotides of the mRNA. The 5'-terminal sequence of the mRNA, absent in the cDNA clone, has been determined with a stretch of cDNA sequence by the primer extension procedure. The amino acid sequence deduced from the nucleotide sequence is consistent with the amino acid sequences of several tryptic peptides, the total amino acid composition, and the mol. wt. of delta-crystallin estimated by SDS-polyacrylamide gel electrophoresis. The computer-assisted analysis predicts high alpha-helical content throughout the polypeptide. Sequence analyses have revealed that gene 1 encodes the mRNA from which the cDNA clone was derived.