Biomedical subjects
P J Bruns
Publications and source records attributed to P J Bruns.
Biolistic transformation of macro- and micronuclei.
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A mutational analysis of conjugation in Tetrahymena thermophila. 1. Phenotypes affecting early development: meiosis to nuclear selection.
Conjugation in the freshwater ciliate Tetrahymena thermophila involves a developmental program that models meiosis, fertilization, and early developmental events characteristic of multicellular eukaryotes. We describe a gallery of five early-acting conjugation mutations. These mutants, cnj1-5, exhibit phenotypes in which specific steps in the conjugal pathway have been altered or eliminated. Specifically, cnj1 and cnj2 fail to condense their micronuclear chromatin prior to each of the three prezygotic nuclear divisions. This results in nuclear division failure, failure to replicate DNA, and failure to initiate postzygotic development. The cnj3 mutant appears to exhibit a defect in chromosome separation during anaphase of mitosis. cnj4 mutants successfully carry out meiosis I, yet are unable to execute the second meiotic division and abort all further development. cnj5 mutants are unable to initiate either meiosis I or meiosis II, yet proceed to execute all subsequent developmental events. These mutant phenotypes are used to draw inferences regarding developmental dependencies that exist within the conjugation program.
Germline and somatic transformation of mating Tetrahymena thermophila by particle bombardment.
Mating Tetrahymena thermophila were bombarded with ribosomal DNA-coated particles at various times in development. Both macronuclear and micronuclear transformants were recovered. Optimal developmental stages for transformation occurred during meiosis for the micronucleus and during anlagen formation for the macronucleus. Evidence is given for transient retention of the introduced plasmid. Genetic and molecular tests confirmed that sexually heritable transformation was associated with integration at the homologous site in the recipient micronuclear chromosome.
A method for mapping germ line sequences in Tetrahymena thermophila using the polymerase chain reaction.
A method for mapping DNA sequences to specific germinal chromosomes in the ciliated protozoan Tetrahymena thermophila has been developed. This mapping technique (PCR mapping) utilizes the polymerase chain reaction and template DNA derived from nullisomic strains to directly assign micronuclear DNA sequences to specific micronuclear chromosomes. Using this technique, a number of unique sequences and short repetitive sequences flanked by unique sequences have been mapped to four of the five germinal chromosomes.
Mapping the mating type locus of Tetrahymena thermophila: meiotic linkage of mat to the ribosomal RNA gene.
Tetrahymena thermophila has a multiple mating type system. While a sexually mature cell usually expresses only one mating type, its germline (micronucleus) carries the genetic potential for 5 to 7 mating types. The set of allowed mating types is specified by the mat locus. The choice of which particular mating type is expressed by a cell reflects a somatically inherited, developmentally programmed differentiation of the somatic nucleus (macronucleus). In this work we report that the mat locus maps to the left arm of chromosome 2, as determined by nullisomic deletion mapping. We also report a distance of 29 cM between the mat locus and the ribosomal RNA gene, previously mapped to chromosome 2L. This represents another (rare) case of meiotic linkage in Tetrahymena.
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.
Phenotypic assortment in Tetrahymena thermophila: assortment kinetics of antibiotic-resistance markers, tsA, death, and the highly amplified rDNA locus.
Phenotypic assortment in Tetrahymena thermophila results from random distribution of alleles during amitotic division of the macronucleus. The rate of assortment is dependent on input ratio and the number of assorting units. The assortment of the antibiotic resistance markers Chx, Mpr and gal was determined and is consistent for each with the model of 45 assorting chromosomes. The gene tsA (previously ts-1) shows normal assortment, in contrast to previous reports. A mutation in the highly amplified ribosomal locus (rdnA2) assorts as if present at only 45 copies. Death of clones occurred at a rate consistent with assortment for a single gene.
A conditional mutant having paralyzed cilia and a block in cytokinesis is rescued by cytoplasmic exchange in Tetrahymena thermophila.
Nineteen mutants that are conditional for both the ability to regain motility following deciliation and the ability to grow were isolated. The mutations causing slow growth were placed into five complementation groups. None of the mutations appears to affect energy production as all mutants remained motile at the restrictive temperature. In three complementation groups protein synthesis and the levels of mRNA encoding alpha-tubulin or actin were largely unaffected at the restrictive temperature, consistent with the hypothesis that mutations in these three groups directly affect the assembly of functional cilia and growth. Complementation group 1 was chosen for further characterization. Both phenotypes were shown to be linked, suggesting they are caused by a single mutation. Group 1 mutants regenerated cilia at the restrictive temperature, but the cilia were nonmotile. This mutation also caused a block in cytokinesis at the restrictive temperature but did not affect nuclear divisions or DNA synthesis. The block in cell division was transiently rescued by wild-type cytoplasm exchanged when mutants were paired with wild-type cells during conjugation (round 1 of genomic exclusion). Thus, at least one mutation has been isolated that affects assembly of some microtubule-based structures in Tetrahymena (cilia during regeneration) but not others (nuclei divide at 38 degrees), and the product of this gene is likely to play a role in both ciliary function and in cytokinesis.
Tetrahymena conjugation-induced genes: structure and organization in macro- and micronuclei.
The physical organization of eight Tetrahymena genes active during conjugation (meiosis) was examined in the somatic (macro-) and germinal (micro-) nuclei of this organism. Three of these genes make transcripts that are only detected during meiotic prophase. Southern blot analyses indicated that all genes examined were present in both nuclei. Except for one gene family (pC6), all appeared to be non-repetitive and there were no detectable sequence rearrangements in or near the genes. The exceptional gene was repeated approximately five to seven times and DNA rearrangement occurred in or near each of these copies. A comparison of cDNA and macro- and micronuclear DNA restriction maps indicated that one of the genes (cnj B) contains introns. This is the first report of evidence for introns in a non-ribosomal gene in Tetrahymena. A site specific modification probably due to adenine methylation was seen in the macronuclear copy of another gene (cnj C).
A drug-resistant mutation in the ribosomal DNA of Tetrahymena.
A mutation that confers resistance to the drug paromomycin is shown to be in the structural gene that codes for the ribosomal RNA in Tetrahymena. This observation was made by exploiting a variant of the ribosomal DNA that distorts amplification of this locus when a new somatic nucleus develops during conjugation. Because the allelic forms of this locus have a restriction endonuclease site polymorphism, it was possible to correlate drug resistance with presence of a specific allele. The genetic results have been confirmed by sequence analysis (presented elsewhere). Thus, the crosses presented here provide a unique opportunity to identify mutations in the ribosomal DNA.
RNA and protein synthesis during meiotic prophase in Tetrahymena thermophila.
Tetrahymena is one of the few organisms from which large amounts of precisely staged meiotic material can be obtained. We took advantage of this fact to monitor RNA and protein synthesis during meiosis. The rate of total protein synthesis as well as the synthesis of the majority of heavily labeled conjugation-specific polypeptides (monitored by high resolution two-dimensional gel electrophoresis) was maximal during meiotic prophase. We therefore cloned cDNAs corresponding to genes active during this time. The mRNA levels of three conjugation-specific genes (pC1, pC2, and pC7) and one conjugation-induced gene (pC3) were followed by using the corresponding labeled cDNAs to probe RNA isolated from different times during mating that was also followed cytologically. Synthesis of the conjugation-specific mRNAs was maximal just prior to maximum crescent stage (pachytene). Evidence is presented for transcription by the normally inactive micronucleus just prior to the maximum crescent stage, confirming an earlier report. The significance of these results is discussed.
Chromosome-designated mutation selection in Tetrahymena thermophila.
Two protocols are presented that allow the selection of mutations mapping to micronuclear chromosome 5 in Tetrahymena thermophilia. One protocol involves crossing mutagenized diploid cells directly to a strain nullisomic for chromosome 5 and screening the monosomic progeny for a mutant phenotype. The second protocol first takes the mutagenized diploid cells through round I of genomic exclusion to create useful and reusable mutant heterokaryons, which are then assayed for the presence of mutations on chromosome 5 by crossing to the nullisomic 5 strain. Of 14 putative chromosome 5 mutations obtained by these two methods, seven are shown by genetic analysis to be recessive mutations on chromosome 5; one mapped elsewhere in the genome; six were infertile or failed to yield progeny in some of the diagnostic crosses and, thus, their genetic nature could not be determined with certainty.
Cloning of abundant mRNA species present during conjugation of Tetrahymena thermophila: identification of mRNA species present exclusively during meiosis.
A cDNA library was constructed by using as a template the RNA present during the meiotic prophase of Tetrahymena thermophila. Clones containing cDNA sequences homologous to moderately abundant to abundant transcripts were detected by colony hybridization and confirmed by hybridizing purified cDNA plasmids on filters with labeled RNA probes. Eighteen clones were isolated, and the sizes of their cDNA inserts were determined. Cross-hybridization of individual cDNA plasmid pairs showed that each of these clones contained cDNA that was homologous to one of eight different RNA transcripts. The sizes of the eight RNA transcripts and the stages of the T. thermophila life cycle during which they were present were determined by hybridizing nick-translated cDNA probes against denatured, electrophoresed RNA from various stages. Clones were identified that contained sequences homologous to RNAs present only in early conjugation (meiosis); other clones contained sequences homologous to RNAs which were abundant during conjugation but present at other stages as well. One clone contained a sequence homologous to an RNA that was abundantly present only in nongrowing cells.
Conditional lethality associated with macronuclear development in Tetrahymena thermophila.
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The pre-pairing events in Tetrahymena. II. Partial loss of developmental information upon refeeding starved cells.
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Conjugation in Tetrahymena thermophila. A temporal analysis of cytological stages.
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