the stability in various detergents of transferrin-transferrin receptor complexes from reticulocyte plasma membranes.

Transferrin-membrane protein complexes were solubilized either with 0.4% sodium dodecyl sulfate (SDS), 1% Triton X-100 or 0.5% sulfobetaine 3-14 from the plasma membranes of rabbit reticulocytes previously labeled with 125I and then incubated with 131-labeled transferrin. When the solubilized membranes were analyzed by gel filtration fractionation, marked variation in the preservation of transferrin-transferrin receptor interaction was noted between the three detergents. After SDS solubilization, more than 80% of the 131I-labeled transferrin remained associated with membrane proteins with apparent molecular weight of the transferrin-receptor complexes of 1400 000 and 240 000. In contrast, after Triton X-100 solubilization only 40% of the transferrin was still complexed to membrane proteins with an apparent molecular weight of the complex of 450 000. Dissociation of transferrin from its receptor was most marked following sulfobetaine solubilization, with less than 30% of the transferrin still complexed. Following gel filtration 131I-labeled transferrin-125I-labeled membrane protein complexes were immunoprecipitated with goat specific anti-rabbit transferrin antibodies. The immunoprecipitates were analyzed under stringent dissociating conditions by two SDS-polyacrylamide gel electrophoretic techniques. In a linear 5-25% polyacrylamide gradient the 125I-labeled receptor obtained after membrane solubilization with all three detergents had an apparent molecular weight of 80 000. In contrast, in a different system using 10% polyacrylamide gel two 125I-labeled receptor components were detected wih apparent molecular weights of 90 000 and 80 000. These results demonstrate that estimates of the molecular weight of the transferrin receptor depended on the conditions of electrophoresis and suggest that the transferrin receptor is partially modified, perhaps by glycosylation.

Uniparental cytogamy: a novel method for bringing micronuclear mutations of Tetrahymena into homozygous macronuclear expression with precocious sexual maturity.

A new method of inducing self-fertilization, uniparental cytogamy, yields homozygous germinal and somatic genotypes in the ciliate Tetrahymena thermophila. Progeny are highly fertile and show a marked tendency for precocious sexual maturity. This method is highly effective in protocols designed to generate and express nonlethal dominant or recessive mutations.

The transition from conjugal development to the first vegetative cell division is dependent on RAD51 expression in the ciliate Tetrahymena thermophila.

Rad51p, the eukaryotic homolog of the prokaryotic recA protein, catalyzes strand exchange between single- and double-stranded DNA and is involved in both genetic recombination and double-strand break repair in the ciliate Tetrahymena thermophila. We have previously shown that disruption of the Tetrahymena RAD51 somatic macronuclear locus leads to defective germline micronuclear division and that conjugation of two somatic rad51 null strains results in an early meiotic arrest. We have constructed Tetrahymena strains that are capable of RAD51 expression from their parental macronuclei and are homozygous, rad51 nulls in their germline micronuclei. These rad51 null heterokaryons complete all of the early and middle stages of conjugation, including meiosis, haploid nuclear exchange, zygotic fusion, and the programmed chromosome fragmentations, sequence eliminations, and rDNA amplification that occur during macronuclear development. However, the rad51 null progeny fail to initiate the first vegetative cell division following conjugal development. Coincident with the developmental arrest is a disproportionate amplification of rDNA, despite the maintenance of normal total DNA content in the developing macronuclei. Fusion of arrested rad51 null exconjugants to wild-type cells is sufficient to overcome the arrest. Cells rescued by cytoplasmic fusion continue to divide, eventually recapitulating the micronuclear mitotic defects described previously for rad51 somatic nulls.

RAD51 is required for propagation of the germinal nucleus in Tetrahymena thermophila.

RAD51, the eukaryote homolog of the Escherichia coli recA recombinase, participates in homologous recombination during mitosis, meiosis, and in the repair of double-stranded DNA breaks. The Tetrahymena thermophila RAD51 gene was recently cloned, and the in vitro activities and induction of Rad51p following DNA damage were shown to be similar to that of RAD51 from other species. This study describes the pattern of Tetrahymena RAD51 expression during both the cell cycle and conjugation. Tetrahymena RAD51 mRNA abundance is elevated during macronuclear S phase during vegetative cell growth and with both meiotic prophase and new macronuclear development during conjugation. Gene disruption of the macronuclear RAD51 locus leads to severe abnormalities during both vegetative growth and conjugation. rad51 nulls divide slowly and incur rapid deterioration of their micronuclear chromosomes. Conjugation of two rad51 nulls leads to an arrest early during prezygotic development (meiosis I). We discuss the potential usefulness of the ciliates' characteristic nuclear duality for further analyses of the potentially unique roles of Tetrahymena RAD51.

Characterization of the microheterogeneity of recombinant primate prolactin: implications for posttranslational modifications of the hormone in vivo.

Recombinant baboon and monkey prolactins were expressed in murine C127 cells. The hormones were purified from the conditioned media of these cells using a combination of cation, anion, and gel filtration chromatographies. This purification scheme provided approximately a 20-fold purification of the proteins with a 40% cumulative yield. Sodium dodecyl sulfate gel electrophoresis of the purified hormones in conjunction with Coomassie blue staining and immunoblotting procedures revealed three major prolactin-related bands with molecular weights corresponding to Mr 16,000, 23,000, and 27,000. Based on these analyses the samples were judged to be greater than 90% pure. Amino terminal sequence analysis of the purified baboon and monkey hormones provided three distinct prolactin-related sequences for each preparation. The predominant sequence corresponded to the predicted amino terminal sequences of the hormones which began with leucine at position 1. Two minor sequences, individually representing approximately 10-20% of the total population, were also identified; one starting at position 11 and the other at position 133. Carbohydrate compositional analysis of the proteins suggested that greater than 50% of the population were glycosylated with a fucosylated complex oligosaccharide. Analysis of the specific bioactivity of the recombinant hormones in the Nb2 cell proliferation assay showed them to be comparable to the NIH and WHO human pituitary-derived standards.

Studies on the microheterogeneity and in vitro activity of glycosylated and nonglycosylated recombinant human prolactin separated using a novel purification process.

Recombinant human PRL was produced in a murine C127 cell expression system and purified to greater than 97% homogeneity using anion and cation exchange chromatography. This material was biologically equivalent to pituitary-derived PRL in both an enzyme-linked immunosorbent assay and the Nb2 lymphoma cell proliferation assay. The predominant PRL forms were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting as being 23 and 25 kilodaltons (kDa). These mass values were confirmed by electrospray mass spectroscopy. Glycosidase digestions indicated that the 25-kDa PRL is N-glycosylated and sialylated, whereas 23-kDa PRL is nonglycosylated. Glycosylated and nonglycosylated forms of the hormone were individually purified to greater than 95% homogeneity using novel cation exchange chromatography. Isoelectric focusing demonstrated that both forms consist of multiple charge isomers, with the charge heterogeneity of the glycosylated form primarily due to differences in sialylation. Monosaccharide analysis of the glycosylated form suggested a minimal complex oligosaccharide chain that may be fucosylated and partially sialylated. Oligosaccharide mol wt were determined by electrospray ionization mass spectroscopy. Analysis of the oligosaccharides by fluorophore-assisted carbohydrate electrophoresis indicated that bi- and triantennary oligosaccharide forms are predominant and have multiple combinations of terminal sialylation. Both forms of PRL were active in the Nb2 lymphoma cell proliferation assay; however, the 23-kDa nonglycosylated form was 3-4 times more active in this assay than the 25-kDa glycosylated form.

Biological activity and metabolic clearance of a recombinant human thyrotropin produced in Chinese hamster ovary cells.

The presence and specific structures of the oligosaccharides on TSH have been shown to be important for its production and bioactivity. Since the carbohydrate structure of a protein reflects the glycosylation apparatus of the host cells in which the protein is expressed, we examined the biological activity and metabolic clearance of a preparation of purified recombinant human (rh) TSH derived from a stable transfectant of Chinese hamster ovary cells. Carbohydrate compositional analysis of this rTSH showed it to be more highly sialylated than a nonrecombinant, cadaver-derived pituitary hTSH. In addition, no N-acetyl galactosamine was detectable in rhTSH, which implies the absence of terminal sulfate moieties, both of which are present in pituitary-derived TSH. The immunologic activity and porcine TSH receptor-binding activity of the preparation of rhTSH were 3- to 4-fold lower than those of a standard pituitary hTSH. The rhTSH showed a maximum stimulatory activity similar to that of pituitary hTSH in two different in vitro bioassays. However, rhTSH elicited about 3-fold and 5-fold less cAMP than pituitary TSH after stimulation of adenylyl cyclase in bovine thyroid membranes and the rat FRTL-5 cell line, respectively. Removal of sialic acid did not alter the immunologic activity of rhTSH. However, the potencies of rhTSH in receptor-binding, adenylyl cyclase, and FRTL-5 assays were increased 2.4-, 2.6- and 26.7-fold, respectively after sialic acid removal. These data suggest that the in vitro biological activity of rhTSH is influenced by its highly sialylated oligosaccharide chains. The rhTSH had a 2-fold lower metabolic clearance rate than pituitary TSH, resulting in a greater than 10-fold higher serum concentration of rhTSH at 3 h as compared to pituitary hTSH. After sialic acid removal, the rhTSH was cleared faster (7.5-fold) than pituitary hTSH, showing that its longer plasma half-life was due to its higher sialylation. Biologically active rhTSH should be of clinical value in the diagnosis and treatment of patients with thyroid cancer and as a pure hTSH reference preparation.

The Nucleic Acid Blot Analyzer II. ANALYZE, an image analysis software package for molecular biology.

The Nucleic Acid Blot Analyzer, a new instrument providing high-speed imaging of 32P labeled nucleic acids, captures, stores and presents images in digital form, thus lending itself to rapid data handling and analysis as well as replacing conventional X-ray film autoradiography for many applications. A software package called ANALYZE has been specifically designed for the instrument in order to provide automatic or semi-automatic analysis for molecular biological techniques. The software includes image display manipulation, quantitative and positional analysis, as well as file maintenance utilities. The specific application of the software/hardware to various techniques is presented.

Recombinant human thyroid stimulating hormone: development of a biotechnology product for detection of metastatic lesions of thyroid carcinoma.

We have genetically engineered a cell line, and developed a reproducible process, for the expression and purification of biologically active recombinant human thyroid stimulating hormone (rhTSH).rhTSH was expressed by co-transfecting a human alpha-subunit cDNA with a human beta-subunit partial genomic clone into Chinese Hamster Ovary (CHO) cells. Stable transfectants which expressed high levels of rhTSH were selected, and subsequently cultured on microcarrier beads. The rhTSH-containing media, produced under serum-free conditions, was clarified and purified by a combination of ion exchange, dye and gel filtration chromatographies. Individual step recoveries were greater than 90% with the exception of a very conservative pooling of the final gel filtration step (78% recovery) that resulted in a cumulative yield of 54% for the purification process. Purity of the final bulk material was judged to be > 99% by SDS polyacrylamide gel electrophoresis (SDS-PAGE), reverse phase HPLC, and size exclusion chromatography. Initial characterization of the oligosaccharide composition indicated the presence of partially sialylated bi- and triantenary complex oligosaccharides. Purified rhTSH was active in a thyroid membrane bioactivity assay with a specific activity of 8.2 IU/mg. The in vivo activity of rhTSH in cynomolgus monkeys appeared to be equal to or greater than that reported for bovine TSH (bTSH) in human subjects. The rapid clearance phase half-life of rhTSH was approximately 35 minutes while the post-distribution phase half life was approximately 9.8 hours. Furthermore, the monkeys showed cumulative increases in minimum plasma rhTSH levels when given three daily intramuscular (IM) rhTSH injections; a phenomenon not observed when bTSH had been administered to humans. The rhTSH showed no evidence of toxic or adverse effects when administered at doses up to 7.2 IU/kg and 0.52 IU/kg in rat and monkey, respectively. These are 50X and 4X multiples of the bTSH doses of 0.143 IU/kg (10 IU/70kg) previously administered to humans.

Conjugal blocks in Tetrahymena pattern mutants and their cytoplasmic rescue. I. Broadened cortical domains (bcd).

The cortical pattern mutant broadened cortical domains (bcd) in Tetrahymena thermophila is unable to complete the nuclear events associated with conjugation. bcd x bcd pairs become arrested at the "nuclear exchange" configuration. Genetic analysis reveals that the bcd conjugal block is 100% penetrant, under macronuclear control, and rescueable (a) by outcrossing to a wild-type partner, (b) by administration of a hyperosmotic shock 5 hr after cells are mixed for mating, or (c) by cytoplasmic transfusion from a wild-type donor. Cytological analysis reveals that the conjugal block is primarily the result of failure in pronuclear fusion (karyogamy). bcd pairs also exhibit reduced nuclear exchange efficiency and a failure of macronuclear anlagen formation. The hypothesis is proposed that the bcd+ gene codes for a microtubule-based organelle "motor" similar to kinesin.

Biochemical and cytological evidence for an overabundance of mucocysts in the bcd pattern mutant of Tetrahymena thermophila.

Three acidic proteins (42 kD, 43 kD and 50 kD) were present in unusually high concentrations in cortical preparations of the Tetrahymena pattern mutant broadened cortical domains (bcd). Antisera to the 42-kD and 50-kD proteins bound to discharging mucocysts and food vacuole contents in both wild-type and mutant cells. Subsequent analysis revealed that bcd mutant cell pellicles possess five times more "docked" mucocysts than their wild-type counterparts.

Conjugal blocks in Tetrahymena pattern mutants and their cytoplasmic rescue. II. janus A.

A conjugal block phenotype is described for the Tetrahymena pattern mutant, janA. janA exhibits a characteristic "janus" phenotype in which cells develop with a global mirror-image duplication of the ventral pattern of cortical organelles. janA cells are competent to form mating pairs, but later become irreversibly fused as heteropolar doublets. The few pairs that successfully dissociate fail to undergo postconjugal oral replacement and perish. The janA conjugal block is 100% penetrant, is under prezygotic macronuclear control, and is lethal. Here we characterize this conjugal block genetically and cytologically and demonstrate that it can be rescued by a transferable, wild-type product. New insights into late conjugal events, especially the replacement of the oral apparatus, are reported for wild-type cells as well.

The morphology of the central nervous system of the barnacle, Semibalanus cariosus (Pallas).

The central nervous system of the sessile barnacle, Semibalanus cariosus (Pallas), has been studied with the particular aim of determining the locations of neuron somata in relation to peripheral nerves. This was accomplished by tracing peripheral nerves using dissection and methylene blue staining techniques, histological methods, and by permitting cobaltous chloride to diffuse via axons into ganglia ("backfilling"). The neuron maps resulting from the study reveal some well-defined sub-systems, a considerable degree of functional clumping of neuron somata, and some unexpected projections of neurons in the CNS. Neurophysiological studies based on these findings are in progress.

The role of cortical geometry in the nuclear development of Tetrahymena thermophila.

Vegetative cells were subjected to electrofusion and the resulting heteropolar doublets were then mated to normal single cells and followed throughout conjugation using cytological and genetic techniques. The unique cyto-geometry created in a heteropolar doublet--a continuous cytoplasmic compartment bounded by two anterior poles and sharing a fused posterior pole at midbody, and the potential for two conjugal exchange junctions--resulted in instructive perturbations of nuclear behavior. Our results indicate that the course of nuclear development is strongly dependent on the cortical geometry of conjugating cells. Specifically, 1) continuation of development after meiosis requires an established conjugal junction; 2) after pronuclear exchange, pronuclei are subjected to attractive forces; and 3) products of the second postzygotic division are actively positioned near the posterior region of the cell cortex where they develop into micronuclei.

A cytogenetic study of development in mechanically disrupted pairs of Tetrahymena thermophila.

We examined the nuclear behavior of mating Tetrahymena cells that had been mechanically disrupted at various times throughout conjugation. Disruption was achieved by agitating conjugating Tetrahymena in the presence of 0.1-3 mm glass beads. Two minutes of agitation with 1 mm beads yielded optimal pair disruption (70%) with high viability (92%). Disrupting pairs between 0-4.7 h after the initiation of mating produced mostly disrupted conjugants in which development was aborted. However, as many as 20% of these early disrupted conjugants completed development even without their mating partners. After 5 h the percentage of disrupted conjugants completing development increased dramatically, reaching 80% by 6.7 h. These results support a model suggesting that events associated with nuclear exchange and fusion 5 h into conjugation trigger a commitment to completion of the postzygotic developmental program. The early conjugants that completed development following disruption suggest that development can be sustained even in the absence of a mating partner. This represents a novel method of bringing the micronuclear genome into macronuclear expression with minimal cytoplasmic exchange between partners. We discuss these results in light of a model relating cortical and nuclear signaling events that reciprocally drive conjugal development.

The pattern of campaniform sensilla on the wing and haltere of Drosophila melanogaster and several of its homeotic mutants.

A detailed mapping and description of campaniform sensilla on the wing and haltere of Drosophila melanogaster is provided. Six types of sensilla are distinguished. Similarities in the pattern of their distribution on the dorsal and ventral surfaces of each appendage, as well as between the wing and haltere, are apparent. These data are used to assess the quality of homeotic transformation in several mutants of the bithorax complex in which the halteres are transformed into wings. Flies homozygous for abxbx3pbx produce a complete inventory of wing sensilla on the homeotic appendage. In abx, bx3 and bx3pbx homozygotes the transformation of haltere into wing is incomplete, and each mutant shows characteristic fields of haltere and wing sensilla. It appears that specific regions of the anterior haltere compartment require different combinations of mutant alleles to produce a distinct homeotic transformation. Furthermore, the pbx mutation appears to influence expression of the bx3 mutation within the anterior compartment.

A mutational analysis of conjugation in Tetrahymena thermophila. 2. Phenotypes affecting middle and late development: third prezygotic nuclear division, pronuclear exchange, pronuclear fusion, and postzygotic development.

Conjugation following pair formation in Tetrahymena can be divided into three distinct sequences of events: prezygotic development, postzygotic development, and exconjugant development. The decision to proceed with postzygotic development is governed by a developmental checkpoint occurring sometime during the middle stages of conjugation. A second developmental decision is made to initiate pair separation and exconjugant development. This paper examines the phenotypes of five newly isolated conjugation mutants (cnj6-cnj10) which affect middle and late events within the conjugation program. cnj6 mutants exhibit normal nuclear behavior throughout development up to and including differentiation of new macronuclear anlagen. Pairs arrest at this developmental endpoint, unable to dissociate. cnj7 and cnj8 eliminate the third prezygotic nuclear division and the first postzygotic nuclear division. All subsequent developmental events appear normal. cnj9 eliminates the second postzygotic nuclear division, and subsequently, new macronuclei fail to develop despite parental macronuclear degradation. cnj10 results in a pleiotropic phenotype characterized by failure of numerous events which all appear to involve nuclear-cytoskeletal interactions. These defects include nuclear selection (anchoring nuclei to the exchange junction), pronuclear exchange, pronuclear fusion, and anchoring postzygotic nuclear division products to the posterior cell cortex. These mutant phenotypes are used to draw inferences regarding developmental dependencies that govern a cell's entry into the postzygotic and exconjugant developmental programs.

Cytosol intermediates in the transport of iron.

Three 59Fe-labeled nonheme components of the cytosol were identified when rabbit reticuloyctes were incubated with 59Fe-labeled plasma under conditions in which the iron supply was not limiting. Two of these components were identified as ferritin and transferrin. The latter was characterized by gel filtration as having apparent molecular weight higher than transferrin, indicating that the transferrin may be complexed to another moiety. The third component, referred to as iron-binding protein-I (IBP-I), is as yet uncharacterized. When the reticulocytes were incubated with unlabeled plasma after pulse-labeling with 59Fe-labeled plasma, 59Fe radioactivity in these cytosol components decreased; after 15 min of chase, the 59Fe in ferritin, transferrin, and IBP-I fell to 64.6%, 26.5%, and 65.8% of the initial values, respectively. A good correlation existed between the decrease of 59Fe in these three nonheme compartments and the associated increase in 59Fe-heme. The data presented suggest that cytosol ferritin, transferrin, and IBP-I are intermediates in the transport of 59Fe from the plasma membrane to the mitochondria.

Development in electrofused conjugants of tetrahymena thermophila.

Electric shock can create parabiotic fusions of living Tetrahymena cells. In this study, cells were mated and successful pairs were electrofused with either vegetatively growing cells or other mating pairs. In particular, we electrofused pairs from normal [diploid x diploid] matings with vegetatively dividing cells in G- or M-phase of the cell cycle. We also fused [diploid x diploid] conjugants with mating pairs involving an aneuploid partner [diploid x "star"], which typically undergo an abortive conjugal pathway termed genomic exclusion. Using such parabiotic fusions we identified and characterized two developmentally critical landmarks: 1) the "abort" signal, which is initiated in pairs with nuclear defects (this first becomes evident soon after the completion of Meiosis I or the beginning of Meiosis II); and 2) the "terminal commitment point", a developmental stage in normal [diploid x diploid] pairs after which conjugation no longer responds to a parabiotically transmitted abort signal (this correlates with the onset of the second postzygotic nuclear division). Finally we demonstrate that a conjugal-arrest-activity varies with the vegetative cell cycle, reaching its highest level of activity during M-phase and dropping just after cytokinesis.