Non-Mendelian Inheritance in Blepharisma intermedium

SYNOPSIS. An albino mutation of Blepharisma intermedium (a dark red ciliate) has been isolated. The inheritance of factors controlling pigmentation, mating-type, and lethality level of sexually derived offspring has been studied. All 3 traits are inherited in a non-Mendelian manner, perhaps via the cytoplasm.     

Axenic Media for Blepharisma intermedium

SYNOPSIS. Blepharisma intermedium was cultured axenically. The organisms were freed of contaminating microorganisms by serial washing in a sterile salt solution. The major nutrients in the various media were: Freshly killed or lyophilized and autoclaved bacteria ( Pseudomonas ovalis ), yeast extract prepared from Fleischmann's baking yeast or Fleischmann's yeast autolysate, lettuce infusion or stigmasterol, 6 B-vitamins, and phosphate buffer at 2 times 10⁻³ M. The cultures were kept in the dark at 25 C. Altho the 1st division after transfer into fresh media was delayed, B. intermedium divided in approximately 32 hours thereafter in most of these media.     

Characterization of protein kinases from Blepharisma intermedium.

Three protein kinases (EC 2.7.1.37) were detected in Blepharisma and partially purified. The enzymes were most active with histone as substrate protein. The stability of the bond between phosphate and protein acceptor showed the characteristics of seryl- or threonylphosphate. Protein kinase I was solubilized by ultrasonication or freezing and thawing, while the enzymes II and III were readily solubilized by mild homogenization. Protein II and III were noticeably activated by cAMP and cGMP, while protein kinase I was inhibited by cAMP. Associated with protein kinase II and III activity was the ability to bind labeled cAMP. The following molecular weights were determined: 90000 for enzyme I, 280000 for enzyme II, and 95000 for enzyme III. Various apparent Michaelis constants were estimated.     

Nuclear Activity During Regeneration in Blepharisma intermedium Bhandary*

SYNOPSIS. Transection and regeneration of Blepharisma intermedium initiate complex macronuclear activity and micronuclear division. Reversible condensation of the macronucleus is achieved by contraction which involves primary and secondary coiling. Primary coiling, evident in vegetative cells, is enhanced by pronounced constriction of the macronuclear extremities during regeneration. Secondary coiling is restricted to the period of contraction and is initiated at the ends of the mauonucleus. Condensation terminates the coiling processes and gives rise to an amorphous structure. Re-elongation of the macronucleus is not followed immediately by coiling. Micronuclear division begins early in regeneration, but the peak of visible activity is not reached until the 3rd through 5th hours.     

Cytochemical Analysis of Enzyme Activity during Regeneration in Blepharisma intermedium Bhandary

SYNOPSIS. Transection and repair in Blepharisma intermedium modify the distribution of several enzymes. The number of active phagosomes is reduced and there are increased staining reactions for acid phosphatase, the nucleases and type-B esterases in the ground cytoplasm. 5'-Nucleotidase activity is localized in the cytoplasmic matrix. Except for ribonuclease, the enzymes may have a perimacronuclear deposition at some period during regeneration. Acid phosphatase may be involved in development of the oral membranelles. Adenosine triphosphatase and lactic dehydrogenase have a moderate staining intensity whereas malic and succinic dehydrogenases have a strong reaction in the cytoplasm; in addition, these enzymes may be localized in the kinetics involved in the neoformation of the oral concrescence. Nuclear reorganization is attended by an increased frequency of peripheral dehydrogenase activity in the late stages of regeneration. These observations are compared with catalytic systems known to function in protozoan metabolism and in vertebrate regeneration.     

DNA-Dependent RNA Synthesis in Isolated Macronuclei of Blepharisma intermedium (Ciliata)

SYNOPSIS. Autoradiographic studies on DNA-dependent RNA synthesis in isolated macronuclei of Blepharisma intermedium in vitro are described. Isolated macronuclei incubated in a medium containing RNA precursors incorporated cystidine and uridine into RNA. Uptake of cystidine was greater than that of uridine. Addition of ATP did not appreciably affect the uptake of precursors into RNA. Actinomycin D (5 μg/ml) blocked incorporation of cystidine and uridine. It is concluded that in isolated macronuclei of Blepharisma , RNA precursors are 1st converted into triphosphates and then incorporated into RNA thru DNA-dependent RNA polymerase.     

Observations on the Fine Structure of the Nuclear Apparatus of Blepharisma intermedium Bhandary (Ciliata: Spirotricha)

SYNOPSIS. An electron-microscope study of the macronucleus and the micronucleus of Blepharisma intermedium Bhandary has been made. Sections show that the macronucleus is bounded by a double membrane. Inside, there are two types of bodies: (a) small irregular bodies, from 0.05 to 0.2 μ in diameter, and (b) larger bodies, from 0.4 to 0.6 μ in diameter. The former are intrepreted as cut ends of long, branching filaments traversing the nuclear cavity in all directions. They correspond to the DNA filaments obtained by centrifugation and KCN action on the macronucleus. Each filament is made of fibrils aboue 150 Å thick. The large bodies correspond to the nucleoli; they also show a fibrillar structure. They offer the added interest of displaying dense particles, from 100 to 800 Å in size, whose nature and significance are obscure. The micro-nucleus has a double membrane, and the contents are divisible into an electron-dense network and a material of low density which fills the interstices of the network.     

Cell surface control in Blepharisma intermedium (Protozoa ciliata). Blocking of gamone II-induced homotypic pairing by different membrane interacting ligands

PHA, Con-A, or anti-tubulin antibodies inhibit homotypic pair formation, in B. intermedium mating type-I cells in the presence of suboptimal concentrations of gamone II. The inhibition is dependent on the dose of gamone added; the structural conformation and the relative concentration of the inhibitor; and the time of addition of the inhibitor. The block can be selectively prevented by competitive inhibitors of each ligand. The receptors for the inhibitors are distinctive and there is no cross-reaction between the ligands. It is concluded that ligand binding and subsequent receptor-ligand aggregation must induce a change within the cell-surface membrane, which distorts the distribution and/or affects an optimal conformational aspect of a specific membrane-receptor system for the gamone, a prerequisite for cell pair formation.     

Taxonomy of Genus Blepharisma with Special Reference to Blepharisma undulans

SYNOPSIS. Three new species of Blepharisma are presented: B. seshachari sp. nov., B. intermedium sp. nov., and B. tropicum sp. nov. Two sub-species of Suzuki are elevated to specific rank, B. undulans americanum to B. americanum and B. undulans japonicum Suzuki to B. japonicum Suzuki. Stein's taxa for the species Blepharisma undulans are considered to be diagnostic for that species.
Important features of morphology and life-cycle of the above mentioned species are given. Discussion and position of the species Blepharisma undulans are presented and a proposal for the reorganization of the species is made. The possibility of a new key derived from a hypothesis of the phylogeny on an evolutionary basis of the genus is presented. This hypothesis has been extended to include ciliates like Stentor and Spirostomum and its implications discussed.