Biomedical subjects

J W Jaraczewski

Publications and source records attributed to J W Jaraczewski.

5 recordsLinked to original sources

PubMed1994

Sequence of the macronuclear DNA encoding large subunit ribosomal protein 29 (L29) in Euplotes crassus and cycloheximide sensitivity.

As a first step towards developing a DNA transformation method for the ciliated protozoan Euplotes crassus we determined the minimum inhibitory concentration (MIC) for cell division in the presence of cycloheximide (Chx) for several cell lines and the range of Chx sensitivity for 106 different progeny cell lines derived by mating two lines. All of the cell lines are highly sensitive to Chx. Progeny cell lines show a wider range of sensitivities than the parental lines. Because site-directed mutagenesis of the RPL29 gene encoding the large subunit ribosomal protein 29 (L29) has been used to generate a Chx-resistance marker (ChxR) for another ciliate, Tetrahymena thermophila [Yao and Yao, Proc. Natl. Acad. Sci. USA 88 (1991) 9493-9497], we isolated and sequenced the entire E. crassus macronuclear DNA carrying RPL29. The encoded peptide is 52-73% identical in sequence to L29 sequences from organisms ranging from T. thermophila and Saccharomyces cerevisiae to mouse. In E. crassus, the codon that has been mutated to confer Chx resistance in both S. cerevisiae and T. thermophila already encodes the amino-acid residue of one of the mutant forms identified in these other organisms. Thus, E. crassus RPL29 is not a convenient source of a selectable marker. Notable features of the macronuclear DNA carrying RPL29 are its extremely short non-coding regions and a TAG stop codon.

Amino Acid Sequence↗

PubMed1994

Developmentally regulated, low abundance Tec element transcripts in Euplotes crassus--implications for DNA elimination and transposition.

During macromolecular development in the ciliated protozoan, Euplotes crassus, > 105 Tec elements are precisely eliminated from the genome in a 2-4 h time interval, generating extrachromosomal circular forms of the elements. Various models have proposed a transposition-based mechanism for this excision. We have tested this hypothesis by determining the abundance of transcripts of Tec element open reading frames (ORFs) and the timing of their appearance. Transcripts are very low in abundance and are only detected by PCR amplification techniques. Thus, the low levels of transcripts argue against the participation of element-encoded functions in the Tec element elimination process. The element transcripts are only detected in RNA samples from mated cells, indicating that the micronucleus and/or developing macronucleus are transcriptionally active during the sexual phase of the life cycle. The transcription detected could allow a low level of germline-specific transposition for these elements.

Animals↗

PubMed1994

Characterization of transcription initiation, translation initiation, and poly(A) addition sites in the gene-sized macronuclear DNA molecules of Euplotes.

The DNA in the transcriptionally active macronucleus of the hypotrichous ciliate Euplotes crassus exists as short, linear molecules with each molecule encoding a single genetic function. Previous work has indicated that coding regions occupy the majority of macronuclear DNA molecules. In the present study we have defined the transcription initiation sites and poly(A) addition sites for a number of different macronuclear genes in Euplotes crassus. Our results indicate that mature transcripts represent all but approximately 100-200 bases of the non-telomeric sequences in macronuclear DNA molecules. We have also examined the sequences in the vicinity of transcription start sites, poly(A) addition sites, and translation initiation sites for Euplotes species genes in an attempt to define the cis-acting elements that control these processes. Our results indicate that some of the common sequence elements known to control these processes in higher eukaryotes are likely not utilized by Euplotes genes. The data do indicate the presence of other conserved sequences both preceding and at the site of poly(A) addition, as well as at the site of translation initiation. These conserved sequences may serve an analogous role in these organisms. Finally, we have found that most macronuclear DNA molecules have transcription initiation sites within 30 bp of the telomere, suggesting that the telomere may play a role in promoting transcription.

Animals↗

PubMed1993

Structures of the Euplotes crassus Tec1 and Tec2 elements: identification of putative transposase coding regions.

The Tec1 and Tec2 transposon-like element families of Euplotes crassus are highly unusual in that all 30,000 copies of each family are excised from the genome during a discrete time period of macronuclear development. Complete nucleotide sequences were generated for the Tec1-1 and Tec2-1 elements, representing the Tec1 and Tec2 families. Open reading frames (ORFs) are conserved in position and sequence between the two elements, although sequences that comprise one ORF (ORF2) of Tec1-1 are split into two overlapping ORFs (ORFs 2A and 2B) in Tec2-1. ORF1 in Tec1-1, its homolog in Tec2-1 and one of the overlapping ORFs from Tec2-1 (ORF2B) contain TGA codons, which may be translated as Cys, as observed for two other Euplotid genes. Sequence analyses of ORFs from other members of each element family indicate that the families are distinct from each other and are highly conserved within each family. Computer searches of sequence databases have revealed sequence similarity between Tec ORF1s and the previously described Tc1-IS630 family of transposases which includes ORFs from bacterial, nematode and insect transposons.

Amino Acid Sequence↗

PubMed1993

Elimination of Tec elements involves a novel excision process.

Approximately 60,000 transposon-like elements of the Tec1 and Tec2 families excise en masse from the micronuclear genome during formation of a macronucleus in Euplotes crassus. The circular product has been shown previously to contain the element inverted repeats joined head to head. To elucidate the mechanism of Tec excision, we have further characterized the circular products. DNA sequence analysis of cloned inverted repeat junctions and of population of supercoiled Tec circles shows that the inverted repeat junctions consist of both copies of the target site duplication surrounding 10 additional bases. The 10 bp differs for each junction. We demonstrate that the circles are highly sensitive to S1, mung bean and Bal 31 nucleases, and the site of sensitivity maps to the junction. Alkaline gel electrophoresis indicates that the junction does not contain a nick or gap; thus, a likely explanation for the nuclease sensitivity is the existence of a heteroduplex DNA structure at the junction. On the basis of these results, we present a model of Tec excision and discuss the relationship of Tec excision to IES elimination and chromosome fragmentation in E. crassus.

Animals↗