Temperature gradient chamber for relative growth rate analysis of yeast.

Relative growth is often used as a phenotypic measure to distinguish mutant and wild-type yeast or bacterial strains. Differential growth as a function of temperature is a convenient and accurate means of analyzing differences between strains. Slight differences in the genotypes of two strains frequently result in differential growth of the two strains as a function of temperature. We have developed a chamber for the simultaneous growth of multiple strains in microtiter plates along a temperature gradient. Image analysis was used to determine colony area and number at various times as a function of temperature. This chamber reduces the time required and increases the accuracy in measuring growth as a function of temperature. This occurs by allowing relative growth to be measured along a temperature gradient where all other conditions are constant. Two strains of yeast (Saccharomyces cerevisiae) with a known difference in temperature dependence of growth were used to demonstrate the performance of this chamber.

Expression of Tetrahymena histone H4 in yeast.

Histone H4 is one of the most conserved proteins known. The very low rate of nonsynonymous substitution in H4 suggests that it fulfills an essential function in virtually all eukaryotes. While the majority of histone H4 sequences differ only slightly from the general consensus H4 sequence, yeast and Tetrahymena sequences diverge substantially from both the consensus and from each other. This study demonstrates that despite this divergence, when Saccharomyces cerevisiae cells are forced to use the Tetrahymena thermophila histone H4 protein, they are viable although they have a reduced growth rate, are temperature-sensitive relative to wild-type, have a lengthened G2 phase, and show a dramatic repression of mating. An amino acid replacement at position 33 in the protein improves the growth rate of these cells growing at temperatures above 28 degrees C. This replacement changes a proline to a serine and is a further divergence from both the Tetrahymena thermophila and Saccharomyces cerevisiae histone H4 sequences. Thus, the replacement and expression of a non wild-type histone H4 in yeast offers measurable effects on cell growth, identifying amino acids required for optimal yeast functioning.

A computer analysis of primer and probe hybridization potential with bacterial small-subunit rRNA sequences.

Analysis of restriction fragment length polymorphism of bacterial small-subunit (SSU) rRNA sequences represents a potential means for characterizing complex bacterial populations such as those found in natural environments. In order to estimate the resolution potential of this approach, we have examined the SSU rRNA sequences in the Ribosomal Database Project bank using a computer algorithm which simulates hybridization between DNA sequences. Simulated hybridizations between a primer or probe sequence and an SSU rRNA sequence yield a value for each potential hybridization. This algorithm has been used to evaluate sites for PCR primers and hybridization probes used for classifying SSU rRNA sequences. Our analysis indicates that length variation in terminal restriction fragments of PCR products from the SSU rRNA sequences can identify a wide spectrum of bacteria. We also observe that the majority of restriction fragment length variation is the result of insertions and deletions rather than restriction site polymorphisms. This approach is also used to evaluate the relative efficiency and specificity of a number of published hybridization probes.

Molecular technique for rapid identification of mycobacteria.

Identification of mycobacteria through conventional microbiological methods is cumbersome and time-consuming. Recently we have developed a novel bacterial identification method to accurately and rapidly identify different mycobacteria directly from water and clinical isolates. The method utilizes the PCR to amplify a portion of the small subunit rRNA from mycobacteria. The 5' PCR primer has a fluorescent label to allow detection of the amplified product. The PCR product is digested with restriction endonucleases, and an automated DNA sequencer is employed to determine the size of the labeled restriction fragments. Since the PCR product is labeled only at the 5' end, the analysis identifies only the restriction fragment proximal to the 5' end. Each mycobacterial species has a unique 5' restriction fragment length for each specific endonuclease. However, frequently the 5' restriction fragments from different species have similar or identical lengths for a given endonuclease. A set of judiciously chosen restriction enzymes produces a unique set of fragments for each species, providing us with an identification signature. Using this method, we produced a library of 5' restriction fragment sizes corresponding to different clinically important mycobacteria. We have characterized mycobacterial isolates which had been previously identified by biochemical test and/or nucleic acid probes. An analysis of these data demonstrates that this protocol is effective in identifying 13 different mycobacterial species accurately. This protocol has the potential of rapidly (less than 36 h) identifying mycobacterial species directly from clinical specimens. In addition, this protocol is accurate, sensitive, and capable of identifying multiple organisms in a single sample.

A molecular technique for identification of bacteria using small subunit ribosomal RNA sequences.

We have recently developed a novel molecular technique for identification of specific bacterial species within a complex mixture. The technique uses PCR to amplify small subunit ribosomal RNA (SSU rRNA) genes from a mixture of bacteria. One of the PCR primers is labeled with a fluorescent dye to allow detection of the amplified product. The PCR product is then digested with restriction enzymes and a capillary electrophoresis unit equipped with a laser-induced fluorescence detector is employed to analyze the restriction fragments. Only restriction fragments that contain the fluorescent-labeled primer are detected. Generally, the nucleotide sequence of the SSU rRNA genes is unique for each bacterial species. Consequently, the fluorescent-labeled restriction fragments from different bacterial species often have characteristic lengths. Thus, the different fluorescent peaks that appear in a capillary electropherogram correspond to labeled restriction fragments from different bacterial species. This protocol allows us to identify a number of different bacterial species in a complex mixture. Only a minute sample of bacterial DNA and a minimal amount of time (8-10 h) are required for this analysis. The protocol is sensitive, rapid and capable of identifying a broad spectrum of bacterial species.

Transformation of Tetrahymena thermophila by microinjection of a foreign gene.

Tetrahymena thermophila has been transformed to paromomycin-resistant phenotypes by microinjection of an aminoglycoside 3'-phosphotransferase (neo) gene under the control of the T. thermophila histone H4-I promoter. This chimeric neo gene, by itself or on a vector containing a rRNA-encoding DNA (rDNA) origin of replication, transforms T. thermophila. In cells transformed with the rDNA origin vector, the neo gene is usually found integrated into the endogenous rDNA molecules and is present in high copy number. In transformants obtained by microinjecting only the linear chimeric gene, the neo gene is found to have replaced the histone H4-I gene or is found integrated into the 5' flanking region of the H4-I gene. The relative transcript levels of the neo gene in T. thermophila transformed by the linear chimeric gene are much higher than in cells transformed with the vector. The neo gene provides an effective selectable marker for transformation of T. thermophila.

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

The 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 and unusual diversity in histone H4 proteins within the Tetrahymena pyriformis complex.

The 29 species of the Tetrahymena pyriformis complex are morphologically identical while being quite diverse at the molecular level. These species are also diverse relative to other eukaryotes. Phylogenetic relationships within the complex have been difficult to determine, because there are groups of closely related species, as well as individual species, that are highly divergent. We have sequenced portions of two highly conserved histone genes and the more rapidly evolving intergenic region between them. These sequences have been used to construct a phylogeny for the complex. A comparison of the amino-terminal portion of the histone H4 proteins from the species of the complex reveals a high degree of sequence diversity relative to that of other organisms. In contrast, the amino-terminal portions of the histone H3 proteins of the species in the complex are identical to each other. We also find that the pattern of nucleotide substitution in the intergenic region differs from that described for higher eukaryotes.

Variable copy number of macronuclear DNA molecules in Tetrahymena.

In Tetrahymena, the DNA of the macronucleus exists as very large (100 to 4,000-kb) linear molecules that are randomly partitioned to the daughter cells during cell division. This genetic system leads directly to an assortment of alleles such that all loci become homozygous during vegetative growth. Apparently, there is a copy number control mechanism operative that adjusts the number of each macronuclear DNA molecule so that macronuclear DNA molecules (with their loci) are not lost and aneuploid death is a rare event. In comparing Southern analyses of the DNA from various species of Tetrahymena using histone H4 genes as a probe, we find different band intensities in many species. These differences in band intensities primarily reflect differences in the copy number of macronuclear DNA molecules. The variation in copy number of macronuclear DNA molecules in some species is greater than an order of magnitude. These observations are consistent with a developmental control mechanism that operates by increasing the macronuclear copy number of specific DNA molecules (and the genes located on these molecules) to provide the relatively high gene copy number required for highly expressed proteins.

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

The species of the Tetrahymena 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 of G. 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.

Characterization of the promoter region of Tetrahymena genes.

The regions between adjacent histone H3 and H4 genes, as well as portions of the genes, from 22 species of Tetrahymena have been amplified using the polymerase chain reaction and sequenced. Both histone genes are transcribed divergently with initiation occurring within the intergenic region, thus 2 sets of 22 homologous Tetrahymena promoters can be compared. A sequence comparison of these regions reveals a single putative promoter element, with a consensus sequence TATCCAATTCARA, present in front of each gene. This sequence contains a 'CCAAT' box, which also occurs at 8 locations preceding other ciliate genes. No other putative promoter sequences are found in front of these sets of histone genes. Sequences searched for include 'TATA' boxes, 'GC' boxes and other sequences suggested as putative promoter elements for ciliate genes. The coding strand immediately preceding ciliate genes is very high in A content and the consensus sequence at the site of protein synthesis is AAAATGG.

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.

Macronuclear DNA molecules of Tetrahymena thermophila.

The physical organization of the DNA in the macronuclei of Tetrahymena thermophila was investigated by using alternating-orthogonal-field gel electrophoresis. The genome consisted of a spectrum of molecules with lengths ranging from less than 100 to in excess of 1,500 kilobase pairs. There were about 270 different macronuclear DNA molecules, with an average size of about 800 kilobase pairs. Specific genes were mapped and were generally found on macronuclear DNA molecules of the same size in different strains of T. thermophila. This indicates that the molecular mechanisms giving rise to the macronuclear DNA molecules were precise. The fragmentation process that gave rise to macronuclear DNA molecules occurred between 11 and 19 h after the initiation of conjugation.

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.

Characterization of the macronuclear DNA of different species of Tetrahymena.

The macronuclear DNAs from 20 different species of Tetrahymena were characterized using Alternating Orthogonal Field (AOF) gel electrophoresis. Each species has approximately 300 different macronuclear DNA molecules that range in size from about 100-2000 kb pairs. Although the individual macronuclear DNA molecules are not well resolved on an AOF gel, most species have a unique profile of macronuclear DNA. The sequences that hybridize with histone H4 (Tetrahymena) and ubiquitin (yeast) genes were identified on the separated macronuclear DNA molecules of the different species. All species have 2 histone H4 genes located on macronuclear DNA molecules of different sizes. This is consistent with the duplication of the histone H4 gene prior to the speciation events leading to the various species of Tetrahymena. The number and sizes of the macronuclear DNA molecules that hybridize with the ubiquitin probe vary from species to species. A grouping of the different species of Tetrahymena based on this hybridization pattern parallels groupings of the species based on ribosomal RNA sequences and isoenzymes. Some intraspecific variation among different strains of Tetrahymena thermophila was detected using ubiquitin and 5S ribosomal RNA as probes.

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.

Hybridization of nucleic acids directly in agarose gels.

Nucleic acids, both DNA and RNA, separated on agarose gels can be visualized by direct hybridization of the dried gel with appropriate radioactive probes. This method does not involve the transfer of the nucleic acid from the gel. The method requires less manipulation than other procedures; it is extremely rapid, sensitive, and inexpensive. These attributes make this procedure a valuable alternative or supplement to the commonly used methods for visualization by hybridization of nucleic acids separated on agarose gels.

Reorganization of unique and repetitive sequences during nuclear development in Tetrahymena thermophila.

Genomic libraries of macro- and micro-nuclear DNA of the ciliate protozoan Tetrahymena thermophila were constructed in the bacteriophage vector lambda gt WES lambda B. Screening of these libraries with a probe for the repeated hexamucleotide sequence C4A2 showed many phage from the micronuclear library but few if any macronuclear sequences having homology to this probe. This is consistent with C4A2-repeating elements being present predominantly if not exclusively at or near the termini of macronuclear DNA. Sequences flanking C4A2-repeating elements were isolated from a number of purified phage and were used as hybridization probes to restriction endonuclease digested macro- and micro-nuclear DNA. These experiments revealed a repeated sequence family as well as unique sequences present only in micronuclear DNA. The repeated sequence element appears to be dispersed throughout the genome. Phage-containing individual members of this micronucleus limited sequence family were purified from the micronuclear library. Some of these phage contained sequences which hybidized to macronuclear DNA. These fragments therefore contain a "transition" region between micronucleus-limited sequences and sequences present in both nuclei.