Physiological Heterothallism and Sexuality in Euascomycetes: A Partial History

Copyright 1998 Academic Press.     

Design of a minimal polypeptide unit for bacteriochlorophyll binding and self-assembly based on photosynthetic bacterial light-harvesting proteins

We introduce LH1beta24, a minimal 24 amino acid polypeptide that binds and assembles bacteriochlorophylls (BChls) in micelles of octyl beta-glucoside (OG) into complexes with spectral properties that resemble those of B820, a universal intermediate in the assembly of native purple bacterial light-harvesting complexes (LHs). LH1beta24 was designed by a survey of sequences and crystal structures of bacterial LH proteins from different organisms combined with currently available information from in vitro reconstitution studies and genetically modified LHs in vivo. We took as a template for the design sphbeta31, a truncated 31 amino acid analogue of the native beta-apoprotein from the core LH complex of Rhodobacter sphaeroides. This peptide self-assembles with BChls to form B820 and, upon cooling and lowering OG concentration, forms red-shifted B850 spectral species that are considered analogous to native LH complexes. We find that LH1beta24 self-assembles with BChl in OG to form homodimeric B820-type subunits comprising two LH1beta24 and two BChl molecules per subunit. We demonstrate, by modeling the structure using the highly homologous structure of LH2 from Rhodospirillum molischianum, that it has the minimal size for BChl binding. Additionally, we have compared the self-assembly of sphbeta31 and LH1beta24 with BChls and discovered that the association enthalpies and entropies of both species are similar to those measured for native LH1 from Rhodospirillum rubrum. However, sphbeta31 readily aggregates into intermediate higher oligomeric species and further to form B850 species; moreover, the assembly process of these oligomers is not reversible, and they are apparently large nonspecific BChl-peptide coaggregates rather than well-defined nativelike LH complexes. Similar aggregates were observed during LH1beta24 assembly, but these were formed less readily and required lower temperatures than sphbeta31. In view of these results, we reevaluate previous in vitro reconstitution studies and propose alternative templates for new designs.     

“Cryptic” dinucleotide polymorphism in the 3′ region of the factor IX gene shows substantial variation among different populations

Long tandem dinucleotide repeats composed of alternating purines and pyrimidines [RY(i)] are abundant and highly polymorphic. Simple RY(i) are predominately composed of one tandem repeat of a dinucleotide sequence. In contrast, cryptic RY(i [cRY(i)] are composed of multiple short dinucleotide repeats. Herein, we describe the racial distribution of alleles for a polymorphic cRY(i) in the factor IX gene. Allele I is absent in Asians, whereas allele III is rare or absent in Caucasians or blacks. A polymorphic cRY(i) analyzed previously shows even more dramatic variation among racial groups, hinting that a battery of cRY(i) might have utility in assessing the racial origin of a DNA sample.     

Expression of zebrafish fkd6 in neural crest-derived glia

The zebrafish fkd6 gene is a marker for premigratory neural crest. In this study, we analyze later expression in putative glia of the peripheral nervous system. Prior to neural crest migration, fkd6 expression is downregulated in crest cells. Subsequently, expression appears initially in loose clusters of cells in positions corresponding to cranial ganglia. Double labelling with a neuronal marker shows that fkd6-expressing cells are not differentiated neurones and generally lie peripheral to neurones in ganglia. Later, expression appears associated with the posterior lateral line and other cranial nerves. For the posterior lateral line nerve, we show that fkd6-labeling extends caudally along this nerve in tight correlation with lateral line primordium migration and axon elongation. Expression in colourless mutant embryos is consistent with these cells being satellite glia and Schwann cells.     

Photoelectric currents across planar bilayer membranes containing bacterial reaction centers. Response under conditions of single electron turnover.

Light-induced electric current and potential responses have been measured across planar phospholipid membranes containing reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides. Under conditions in which the reaction centers are restricted to a single electron turnover, the responses can be correlated with the light-induced electron transfer reactions associated with the reaction center. The results indicate that electron transfer from the bacteriochlorophyll dimer to the primary ubiquinone molecule, and from ferrocytochrome c to the oxidized dimer occur in series across the planar membrane. Electron transfer from the primary to secondary ubiquinone molecule is not electrogenic.     

Cell cycle phase dependent productivity of a recombinant Chinese hamster ovary cell line

A Chinese Hamster Ovary cell line, CHO1-15₅₀₀, producing recombinant human tissue type plasminogen activator (tPA) via the dihydrofolate reductase (DHFR) amplification system, was studied in batch culture. In this system both DHFR and tPA are under the control of the strong constitutive viral SV40 early promoter. Employing the cumulative viable cell-hour approach, the specific productivity of tPA had maxima in the lag (0.065 pg cell⁻¹ h⁻¹) and early decline (0.040 pg cell⁻¹ h⁻¹) population growth phases. The viable population was assigned into four subpopulations (G1, S, G2/M phase, and Apoptotic cells) using flow cytometric analysis. As expected, intracellular DHFR was maximally expressed during the S cell cycle phase. The production of tPA, however, was found to be a direct linear function of the G1 phase, with a subpopulation specific productivity of 0.080 pg c-h⁻¹. Productivity maxima in the lag and early decline corroborate the flow cytometric data, indicative that this recombinant tPA production occurs primarily in the G1 cell cycle phase, not the S phase. This suggests that endogenous regulatory mechanisms are important controlling influences on the production of recombinant tPA in this ovarian cell line. Productivity from recombinant cell lines cannot be inferred from either the plasmid construct or the host cell alone. Elucidation of the relationship between expression of recombinant protein and the cell cycle phases of the host cell is a major component of the characterization of the animal cell production system. This information facilitates rational process design, including operating mode, modelling and control, and medium formulation.     

Structure of the capsular polysaccharide of Klebsiella serotype K79

The structure of the capsular polysaccharide from Klebsiella K79 was determined by the techniques of methylation, periodate oxidation, beta-elimination, chromic acid oxidation, and partial hydrolysis. N.m.r. spectroscopy (1H and 13C) was used extensively to establish the nature of the anomeric linkages of the polysaccharide and of oligosaccharides derived through degradative procedures. The polysaccharide was found to have the heptasaccharide, "5 + 2" repeating unit: (Formula: see text).