Isolation and characterisation of Chlamydomonas reinhardtii mutants with an impaired CO2-concentrating mechanism.

In order to identify new genes involved in the carbon-concentrating mechanism of Chlamydomonas reinhardtii (Dangeard), high-CO2-requiring mutants were isolated by insertional mutagenesis after transformation of strain CC1618 with a plasmid carrying Arg7 as a selectable marker gene. Six mutants were classified by their growth behaviour under ambient CO2, the affinity of the CO2-concentrating mechanism for inorganic carbon and the expression of known low-CO2-inducible proteins. The mass-spectrometric measurement of carbonic anhydrase activity and CO2/HCO3- transport revealed that four of the mutants are unable to induce a high-affinity carbon-concentrating mechanism. The expression of various carbonic anhydrases and chloroplast inner envelope polypeptides was examined with Western Blots. While three high-CO2-requiring mutants showed abnormal expression patterns, one matched the wild type. With Southern blots the total number and structure of the insertion events were determined to select possible candidates for plasmid recovery. Abnormal structures of thylakoid lamellae traversing the pyrenoid were detected by electron microscopy in some of the high-CO2-requiring mutants. Our characterisations of the insertionally generated mutants revealed phenotypes that have not been published before and therefore might be useful tools to obtain new insights on the molecular background of the CO2-concentrating mechanism and its regulation.

The CO2-concentrating mechanism in the physiological context: lowering the CO2 supply diminishes culture growth and economises starch utilisation in Chlamydomonas reinhardtii.

In a synchronously grown Chlamydomonas reinhardtii (Chlorophyceae) culture the CO2-concentrating mechanism (CCM) was induced by lowering the CO2 level from 4% to 0.036% CO2 (culture HL). The effects of the reduced carbon supply on starch levels were studied over a period of up to 100 h and compared with control cultures kept either at 4% CO2 (culture H) or continuously at ambient air (0.036% CO2, culture L). Lowering the CO2 supply reduced culture growth as estimated by chlorophyll, protein and cell density. The starch level continued to show diurnal variations with an initially reduced rate of starch synthesis at reduced or abolished culture growth. Subsequently, starch maxima and minima increased. After 4 days the resulting pattern for culture HL was similar to that of culture L, which possessed higher minima but identical maxima to culture H. The intracellular starch localisation was examined on electron micrographs. Cell extracts were assayed for ADP-glucose pyrophosphorylase (EC 2.7.7.27) and starch phosphorylase (EC 2.4.1.1) activities. Over the assayed period of 2 days, there was a good correlation between the observed changes in the starch levels and the measured enzyme activities. The rate of CO2-dependent oxygen evolution of culture HL declined from 100% to 60% of the control over the day. This indicates that the diminished or abolished growth and the impairment of starch accumulation upon CO2 depletion are not simply consequences of the lowered level of the substrate CO2. The diminished growth and the peculiar starch accumulation pattern with higher positions of the starch minima in low-CO2 cells are interpreted as economised starch utilisation as long-term aspects of induction of the CCM.

[Adenoma and adenocarcinoma of the iris and ciliary body epithelium in the dog]. / Adenom und Adenokarzinom des Iris-Ziliarkörperepithels beim Hund.

An adenoma and an adenocarcinoma of the anterior uvea in two dogs are described clinically--follow-up included--and ophthalmopathologically. Incidence, therapy, differential diagnosis and prognosis of these epithelial neoplasms in the anterior uvea are discussed.