Dawn B. Marks

Biogenesis of thylakoid membranes with emphasis on the process in Chlamydomonas

Recent results obtained by electron microscopic and biochemical analyses of greening Chlamydomonas reinhardtii y1 suggest that localized expansion of the plastid envelope is involved in thylakoid biogenesis. Kinetic analyses of the assembly of light-harvesting complexes and development of photosynthetic function when degreened cells of the alga are exposed to light suggest that proteins integrate into membrane at the level of the envelope. Current information, therefore, supports the earlier conclussion that the chloroplast envelope is a major biogenic structure, from which thylakoid membranes emerge. Chloroplast development in Chlamydomonas provides unique opportunities to examine in detail the biogenesis of thylakoids.

The pressor response to angiotensin II in patients with low renin essential hypertension.

SUMMARY The etiology of low renin essential hypertension (LREH) has not been established with certainty, but mineralocorticoid excess has been implicated frequently in its pathogenesis. The finding of several investigators of a normal exchangeable sodium space and extracellular fluid volume, however, does not support this hypothesis. To evaluate the possible role of sodium and water retention in LREH, the pressor response to infused angiotensin II (A II) was determined and compared to that of normal subjects and that of subjects with normal renin essential hypertension (NREH). This approach was based on the known suprasensitivity of vascular receptors to A II in situations in which sodium and water compartments are expanded as they are, for example, in proven hypermineralocorticoid states such as primary aldosteronism. In this study, we found that subjects with LREH demonstrated no increased pressor response to graded doses of A II; this suggests that LREH is not primarily mediated by sodium and water retention.

Growth of Unicellular Forms of the Fungus Cordyceps militaris and Analysis of the Chemical Composition of their Walls

SUMMARY: When Cordyceps militaris is grown in shaking culture in certain media, two large unicellular forms are produced. The walls isolated from these are similar both qualitatively and quantitatively except for their mannose content, one form containing almost twice the amount as the other (17.5 versus 9.6%). Glucose, the predominant monosaccharide obtained by acid hydrolysis, accounts for more than half (52 to 60%) of the weight of the walls. The remainder is composed of galactose (6%), hexosamine (6%), lipid (8 to 10%), and protein (8 to 10%). Walls of the unicellular forms contain more glucose and protein than those of the filamentous form, but less hexosamine and lipid. Glusulase releases all of the glucose from walls of the filaments, but only half of the glucose from walls of the unicells.

Proteolytic digestion of histones indicates that the nucleosomes of nuclei and of sheared chromatin are similar

Abstract Calf thymus nuclei were treated with trypsin, chymotrypsin or Pronase, and the rate of digestion of the various histone fractions was determined. The results differed from those obtained by digestion of DNA-free histones with the same set of enzymes but were identical to those obtained by digestion of calf thymus chromatin. Because these enzymes have such different specificities, the results of these digestions indicate that the histone fractions have similar locations in the chromosomal substructures of nuclei and chromatin, i.e. that the structure of the nucleosomes which exist within nuclei is not changed markedly when chromatin is isolated from nuclei by a method which involves shearing.

The use of proteolytic enzymes to determine the location of histones in chromosomal substructures.

Abstract We have observed that three proteolytic enzymes with widely different specificities produce a very similar pattern in terms of the order of digestion of the various histone fractions in chromatin. Histone H2A is most resistant to proteolytic attack by trypsin, chymotrypsin, or Pronase. H2B is next most resistant, followed by H3. Histone H1 is least resistant and is rapidly hydrolyzed by each of these enzymes. The behavior of histone H4 differs for the various enzymes. It is as resistant as H2A to digestion by trypsin and chymotrypsin but is readily hydrolyzed by Pronase. A comparison of the rates of digestion of the various histone fractions in chromatin with the rates in a DNA-free histone mixture and a study of the degradation products which result from digestion indicate that histone conformation and histone-histone and DNA-histone interactions are all involved in protecting histones from attack by proteolytic enzymes. From the results of our studies we have concluded that histones H1 and H3 are located in superficial positions of the chromosomal substructures (or nu bodies) while H2A is buried inside. Since histone H2B is relatively resistant to digestion but more readily degraded in chromatin than in a DNA-free histone mixture, it is difficult to determine its chromosomal location. Histone H4 behaves as if a large portion of the molecule is located in the major groove of the DNA helix.

The presence in the fungus Cordyceps militaris of nuclear proteins resembling histones.

Abstract The typical mammalian histones are not present in the fungus Cordyceps militaris . However, two proteins are consistently found in acid extracts of the nuclear fraction. They are moderately lysine-rich, similar to the histones in molecular weight, but different from any known mammalian histone fraction in mobility on acidic polyacrylamide gels. These acid soluble nuclear proteins of C. militaris differ to some extent from the basic nuclear proteins found in yeasts and the filamentous fungus, Neurospora crassa . Unlike the arginine-rich histone fractions, the moderately lysine-rich histomes are found in both higher and unicellular eukaryotes. It is suggested that the moderately lysine-rich histones form the core which is essential for maintaining the unique subunit structure of chromatin.

Role of the Chloroplast Envelope in Thylakoid Biogenesis

Thylakoid biogenesis in Chlamydomonas reinhardtii y-1 occurred at a linear rate when degreened cells incubated at 38°C were exposed to light. Photosystem 2 (PS2) activity, which was negligible in degreened cells, increased in parallel with chlorophyll (Chl). Kinetic parameters suggest that these PS2 units were larger and more efficient in light capture than those made at 25° C. Membranes made during the initial minutes of greening emanated from the chloroplast envelope. Kinetics of accumulation of chlorophyll a/b-binding (Cab) proteins implied that these proteins were degraded immediately upon import into the plastid unless Chl was available. Molecular modeling studies suggest that Chl is required to convert these proteins from an extended, protease-sensitive conformation into a compact, folded complex. A protease was partially purified that may be involved in degradation of the uncomplexed Cab proteins. These studies support the hypothesis that assembly of thylakoid membranes in this organism occurs in association with the chloroplast envelope.

Aspects of Assembly of Chlorophyll A/B-Protein Complexes

Yellow cells of Chlamydomonas reinhardtii y1 green at 38°C sufficiently rapidly that chloroplast development can be examined kinetically over a time span of minutes (1,2). Chlorophyll (Chl) and the Chl a/b-binding (Cab) polypeptides accumulate at linear rates after exposure to light (3,4). To gain information on the process by which these components come together to form the major light-harvesting Chl a/b-protein complexes, we measured the kinetics of accumulation of the major Cab polypeptides and of the Chls that accumulate during the initial period of greening. We also investigated the difference in accumulation of Cab polypeptides in the light or dark.