Clifford F. Brunk

Assay for nanogram quantities of DNA in cellular homogenates

Abstract The DNA concentration of a crude cellular homogenate can be measured accurately in the nanogram range using the fluorescence enhancement of 4′,6-diamidino-2-phenylindole (DAPI) or bisbenzimidazole (Hoechst H 33258) complexed with DNA. A simple assay has been devised including an internal standard, which allows reliable measurement and compensates for any quenching due to cellular components or buffer. The fluorescence enhancement is highly specific for DNA; no other cell component produces significant fluorescence. The response is linear over a broad dynamic range making the measurement of unknown DNA concentrations convenient.

Prokaryotic Genome Size and SSU rDNA Copy Number: Estimation of Microbial Relative Abundance from a Mixed Population.

A bstractDetermination of the relative abundance of a specific prokaryote in an environmental sample is of major interest in applied and environmental microbiology. Relative abundance can be calculated using knowledge of SSU rDNA copy number, amount of SSU rDNA in the sample, and a weighted average estimate of the genome sizes for organisms in the original sample. By surveying the literature, we provide estimates of genome size and SSU rDNA copy number for 303 and 101 prokaryotes, respectively. This compilation can be used to make reasonable estimates for a wide range of organisms in the calculation of relative abundance. A statistical analysis suggests that no correlation exists between genome size and SSU rDNA copy number. A phylogenetic analysis is used to offer insights into the evolution of both genome size and SSU rDNA copy number.

Comparison of various ultraviolet sources for fluorescent detection of ethidium bromide-DNA complexes in polyacrylamide gels

Abstract Ultraviolet sources with output wavelengths of 254, 300, and 366 nm were compared for detection of ethidium bromide-DNA complexes in acrylamide gels. The 254- and 300-nm sources were both much more sensitive than the 366-nm source. The 254-nm source produced a great deal of photodamage, photonicking and photodimerization, and photobleaching, while the longer wavelength sources cause little damage or bleaching. The 300-nm source is clearly the most suitable source, providing high sensitivity and a relatively low amount of photodamage and photobleaching.

Genome Reorganization in Tetrahymena

Publisher Summary This chapter presents a discussion of the genome reorganization in tetrahymena. In many respects tetrahymena is an ideal organism for molecular biological investigations. The concept of reorganization and elimination of DNA sequences as a developmental mechanism for producing stable changes in cell lineages was a prominent idea during the early part of this century. Genome reorganization as a developmen tal process is now well established, but it is not a major feature of most developmental programs. Among the ciliates, however, genome reorganization is a way of life. These organisms have a dual nuclear system. Each cell possesses one or more micronuclei and one or more macronuclei. The macronuclei are responsible for gene expression during the vegetative growth of the cell and the micronuclei are functional during the sexual phase of its life cycle. As pointed out by Gorovsky, tetrahymena affords a unique opportunity to investigate the functions and interactions of germline and somatic nuclei. In the sphere of the dominant role of genome reorganization in the life cycle of the ciliates, tetrahymena is a logical choice for an in-depth study of this process. Tetrahymena, like the hypotrichs, undergoes substantial genome reorganization during conjugation. The micronuclei of tetrahymena contain five chromosomes, which can be observed during meiosis and cytologically, the chromosomes all appear to be in the same size range. The best characterized case of genome reorganization in tetrahymena is the removal of the rRNA gene from an integrated site in the micronuclear chromosome and its rearrangement into a 21-kb palindrome in the macronucleus.

The nucleotide sequence of the small subunit ribosomal RNA gene from Symbiodinium pilosum, a symbiotic dinoflagellate.

SummaryThe complete sequence of the small subunit ribosomal RNA (SSU rRNA) gene was determined for the symbiotic dinoflagellate Symbiodinium pilosum. This sequence was compared with sequences from two other dinoflagellates (Prorocentrum micans and Crypthecodinium cohnii), five Apicomplexa, five Ciliata, five other eukaryotes and one archaebacterium. The corresponding structurally conserved regions of the molecule were used to determine which portions of the sequences could be unambiguously aligned. Phylogenetic relationships were inferred from an analysis of distance matrices, where pair-wise distances were determined using a maximum likelihood model for transition and transversion ratios, and from maximum parsimony analysis, with bootstrap resampling. By either analytical approach, the dinoflagellates appear distantly related to prokaryotes, and are most closely related to two of the Apicomplexa, Sarcocystis muris and Theileria annulata. Among the dinoflagellates, C. cohnii was found to be more closely affiliated with the Apicomplexa than either P. micans or S. pilosum.

Phylogenetic relationships among Tetrahymena species determined using the polymerase chain reaction.

SummaryThe species of theTetrahymena pyriformis complex present a conundrum with regard to their highly conservative morphology and widely divergent molecular characteristics. We have investigated the phylogenetic relationships among these species using the nucleotide sequences from the histone H3II/H4II region of the genome. This region includes portions of the two histone coding sequences, as well as the intergenic region. The DNA sequences of these regions were amplified by the polymerase chain reaction (PCR) and the sequence of each was determined. Nucleotide substitutions and insertions/deletions within this set of sequences were compared to determine the phylogenetic relationships among the species of the complex. These data yield phylogenetic trees with identical topologies when different tree-building routines are used, indicating that the data are very robust.Glaucoma chattoni was used as an outgroup to root the trees for this analysis. The genome organization ofG. chattoni and the divergence of its histone H3II/H4II region sequence relative to those of the complex clearly indicate that this species has diverged considerably from the complex. These results show that PCR amplification analysis is feasible over considerable evolutionary distances. However, DNA-DNA hybridization may be more useful than sequence analysis in resolving the relationships among the closely related species in the complex.

Mitochondrial proliferation during myogenesis

Abstract The mitochondrial (mt) DNA content of muscle cells increases about 4-fold during myogenesis. This is apparently the result of continued mtDNA replication after the myoblasts become post-mitotic and nuclear DNA synthesis ceases. The rate of mtDNA synthesis in prefusion cells exceeds that necessary for growth, indicating mt turnover. Eventually, the mtDNA synthesis drops to about one third the prefusion rate, leading to a stable mtDNA content in muscle tissue.

Elimination of micronuclear specific DNA sequences early in anlagen development.

After conjugation in Tetrahymena thermophila, the old macronuclei degenerate, and new macronuclei (anlagen) develop. During anlagen development a number of DNA sequences found in the micronuclear genome (micronuclear limited sequences) are eliminated from the anlagen. A cloned copy of a repetitive micronuclear limited sequence has been used to determine the developmental stage at which micronuclear limited sequences are eliminated. DNAs from anlagen of various developmental stages were examined by Southern analysis. It was found that micronuclear limited sequences are present in 4C anlagen and essentially absent in 8C and 16C anlagen. The precipitous loss of these sequences in the 8C anlagen rules out under-replication as the mechanism for the loss and suggests that these sequences are specifically degraded early during anlagen development.

Transformation of Tetrahymena thermophila by electroporation and parameters effecting cell survival

We have successfully transformed Tetrahymena thermophila by electroporation, a process of electrically introducing DNA. The DNA used for transformation contains a mutant ribosomal RNA gene (rDNA) that confers resistance to paromomycin on the transformed cells. This mutant rDNA replicates more rapidly than the endogenous rDNA of the transformed cells so that the mutant rDNA becomes predominant within several generations. This mutant rDNA also carries a restriction polymorphism that readily distinguishes it from the endogenous rDNA of the transformed cells. Substantial nuclease activity is released from the cells during electroporation and must be neutralized in order for transformation to be effective. Cell survival is inversely proportional to the electrical energy dissipated (joules) in the medium. Electroporation is a convenient and effective means of introducing transforming DNA into T. thermophila.

Analysis of nuclei from exponentially growing and conjugated Tetrahymena thermophila using the flow microfluorimeter

Isolated nuclei of Tetrahymena thermophila from both exponentially growing cultures and from cells following conjugation have been analysed using a flow microfluorimeter. The macronuclei from a culture in exponential growth display a single broad distribution of DNA contents, without bimodal character. The micronuclei are virtually all in G2 phase (4C). The mean of the macronuclear DNA distribution is about 12.4 times the micronuclear mean (50C). When cells are starved in preparation for conjugation, the macronuclei DNA content is decreased about 30%, but the distribution remains similar to that of nuclei from a culture in exponential growth. Following conjugation, the macronuclear anlagen develop through a set of relatively synchronous endoreplications. At 12 h after the initiation of conjugation the anlagen are at a 4C stage and at 18 h they are virtually all at a 8C stage. If the culture is refed, anlagen development progresses to a 16C and 32C, but the synchrony is poorly conserved. Cells that are not refed are arrested at the 8C stage and only a fraction of the population ever become mature macronuclei. In general we do not observe distinct peaks of anlagen with DNA contents in excess of 32C. The amitotic division of macronuclei may obscure any endoreplications producing anlagen stages with higher DNA content.