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L A Klobutcher

Publications and source records attributed to L A Klobutcher.

At least 19 recordsLinked to original sources
PubMed2000

A development-specific histone H3 localizes to the developing macronucleus of Euplotes.

During the process of macronuclear development, the ciliate Euplotes crassus undergoes extensive programmed DNA rearrangement. Previous studies have identified a gene, H3(P), that is expressed only during sexual reproduction and is predicted to encode a variant histone H3 protein. In the current study, an antiserum to the H3(P) protein has been generated. The antiserum has been used to demonstrate that H3(P) is maximally expressed during the polytene chromosome stage of macronuclear development. Moreover, H3(P) is localized to the developing macronucleus, but not other nuclei present within the cell. Additional studies indicate that at least one additional variant histone is also present within the developing macronucleus. The results indicate that there are significant changes in nucleosome composition within the developing macronucleus, and provide additional support for the notion that changes in chromatin structure play a role in the DNA rearrangement processes of macronuclear development. genesis 26:179-188, 2000.

Amino Acid Sequence↗
PubMed1999

Characterization of in vivo developmental chromosome fragmentation intermediates in E. crassus.

Ligation-mediated PCR was used to characterize intermediates in the fragmentation/de novo telomere addition process that occurs during sexual reproduction in the ciliate E. crassus. Fragmentation generates ends with 6-base, 3' overhangs that have 5'-phosphate and 3'-hydroxyl groups. These intermediates are detected only during the period of chromosome fragmentation. Fragmentation always occurs at a precise distance from a conserved sequence, the E-Cbs, indicating that it is a key cis-acting element in the process. The results also serve to identify the natural substrate for de novo telomere addition and indicate that telomerase recognizes, and compensates for, partial telomeric repeats at the ends of fragmentation intermediates. Similarities of the Euplotes fragmentation/telomere addition process to the movement of some non-long terminal repeat retrotransposons are discussed.

Animals↗
PubMed1998

Conserved DNA sequences adjacent to chromosome fragmentation and telomere addition sites in Euplotes crassus.

During the formation of a new macronucleus in the ciliate Euplotes crassus, micronuclear chromosomes are reproducibly broken at approximately 10 000 sites. This chromosome fragmentation process is tightly coupled with de novo telomere synthesis by the telomerase ribonucleoprotein complex, generating short linear macronuclear DNA molecules. In this study, the sequences of 58 macronuclear DNA termini and eight regions of the micronuclear genome containing chromosome fragmentation/telomere addition sites were determined. Through a statistically based analysis of these data, along with previously published sequences, we have defined a 10 bp conserved sequence element (E-Cbs, 5'-HATTGAAaHH-3', H = A, C or T) near chromosome fragmentation sites. The E-Cbs typically resides within the DNA destined to form a macronuclear DNA molecule, but can also reside within flanking micronuclear DNA that is eliminated during macronuclear development. The location of the E-Cbs in macronuclear-destined versus flanking micronuclear DNA leads us to propose a model of chromosome fragmentation that involves a 6 bp staggered cut in the chromosome. The identification of adjacent macronuclear-destined sequences that overlap by 6 bp provides support for the model. Finally, our data provide evidence that telomerase is able to differentiate between newly generated ends that contain partial telomeric repeats and those that do not in vivo.

Animals↗
PubMed1997

An unusual histone H3 specific for early macronuclear development in Euplotes crassus.

Characterization of the histone H3 genes of the ciliated protozoan Euplotes crassus indicates that one gene functions only during the sexual phase of the life cycle. Maximum expression of this gene, as judged by transcript accumulation, correlates with DNA replications leading to polytenization of the micronuclear chromosomes before massive DNA elimination, which produces a transcriptionally active macronucleus. Transcripts of the other gene accumulate primarily during vegetative growth and in the sexual phase of the life cycle during replication phases not related to polytenization. Although both histone H3 genes encode proteins that are fairly divergent in sequence at the amino terminus, the meiotic/polytene-specific histone H3 contains two insertions in the amino terminus that increase the size of the protein by 15 amino acids. Analysis of micrococcal nuclease digests of chromatin using hybridization probes specific for micronuclear vs. macronuclear sequences indicates that a change in nucleosomal spacing correlates with the maximal expression of the meiotic/polytene-specific histone H3 gene. Thus, we surmise that this unusual histone H3 may play a key role in targeting DNA sequences for either transcriptional activation and retention in the macronucleus or heterochromatization and elimination.

Amino Acid Sequence↗
PubMed1995

Genetic code deviations in the ciliates: evidence for multiple and independent events.

In several species of ciliates, the universal stop codons UAA and UAG are translated into glutamine, while in the euplotids, the glutamine codon usage is normal, but UGA appears to be translated as cysteine. Because the emerging position of this monophyletic group in the eukaryotic lineage is relatively late, this deviant genetic code represents a derived state of the universal code. The question is therefore raised as to how these changes arose within the evolutionary pathways of the phylum. Here, we have investigated the presence of stop codons in alpha tubulin and/or phosphoglycerate kinase gene coding sequences from diverse species of ciliates scattered over the phylogenetic tree constructed from 28S rRNA sequences. In our data set, when deviations occur they correspond to in frame UAA and UAG coding for glutamine. By combining these new data with those previously reported, we show that (i) utilization of UAA and UAG codons occurs to different extents between, but also within, the different classes of ciliates and (ii) the resulting phylogenetic pattern of deviations from the universal code cannot be accounted for by a scenario involving a single transition to the unusual code. Thus, contrary to expectations, deviations from the universal genetic code have arisen independently several times within the phylum.

Animals↗
PubMed1995

Consensus inverted terminal repeat sequence of Paramecium IESs: resemblance to termini of Tc1-related and Euplotes Tec transposons.

During the formation of a transcriptionally active macronucleus, ciliated protozoa excise large numbers of interstitial segments of DNA (internal eliminated sequences; IESs) from their chromosomes. In this study we analyze the published sequences of 20 IESs that interrupt surface protein genes of Paramecium and identify a consensus inverted terminal repeat. This sequence is similar to the ends of the Tc1-related transposons found in nematodes and other metazoans, as well as to both the ends of the Tec transposons and at least some of the IESs in the distantly related ciliate Euplotes crassus. The results of these analyses bolster previous proposals that IESs were created by transposition.

Animals↗
PubMed1995

Developmentally excised DNA sequences in Euplotes crassus capable of forming G quartets.

Tens of thousands of DNA segments are eliminated by DNA breakage and rejoining events during the formation of a new macronucleus in the hypotrichous ciliated protozoan Euplotes crassus. This study presents evidence for a class of eliminated sequences referred to as telomeric-repeat-like internal eliminated sequences (TelIESs). TelIESs are shorter (< 50 bp) than most previously characterized IESs and their DNA sequences resemble the telomeric repeat sequences of the organism. The TelIESs are excised during the developmental period of chromosome fragmentation/telomere addition, which is later than previously characterized IESs. Additional studies demonstrate that oligonucleotides representing the TelIESs are, like telomeric repeats, capable of forming G-quartet structures in vitro.

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

Circular forms of developmentally excised DNA in Euplotes crassus have a heteroduplex junction.

Extensive DNA elimination via a DNA breakage and rejoining process occurs during macronuclear development in the hypotrich ciliate Euplotes crassus. The excision process involves the removal of short, unique segments of DNA (internal eliminated sequences; IESs) and at least two highly repetitive families of transposon-like elements (Tec elements). Previous studies have demonstrated that circular forms of both IESs and Tec elements are generated following their developmental excision and that some flanking DNA sequences are retained at the circle junctions. In this study we have further analyzed the circle junctions of IESs. Analysis of polymerase chain reaction (PCR) products derived from IES circle junctions indicates that at least two sequence arrangements can be present. The circle junctions contain both of the direct repeats that define the ends of the IES separated by either 2 bp flanking the right end of the IES and 8 bp from the left-flanking region, or 8 bp from the right and 2 bp from the left. Using a method that we have termed "strand-biased PCR," we obtained evidence that the junctions of free circular IESs have a 6-base heteroduplex at their center, such that one strand of the DNA is derived from the left-flanking region of the IES and the other from the right. Models of IES excision are presented that incorporate these results and those of previous studies on the excision process.

Animals↗
PubMed1991

High fidelity developmental excision of Tec1 transposons and internal eliminated sequences in Euplotes crassus.

Following the sexual phase of its life cycle, the hypotrichous ciliate Euplotes crassus transforms a copy of its chromosomal micronucleus into a transcriptionally active macronucleus containing short, linear, gene-sized DNA molecules. Tens of thousands of DNA breakage and joining, or splicing, events occur during macronuclear development. The DNA removed by such events includes transposon-like elements, referred to as Tec1 elements, as well as segments of unique sequence DNA, termed internal eliminated sequences (IESs). Both types of elements are bounded by short direct repeats. In the current study, a polymerase chain reaction (PCR) and DNA sequencing strategy has been used to examine the fidelity of excision of two Tec1 elements and three IESs. In all cases, the vast majority of excision events were found to be precise, with one copy of the terminal direct repeats retained at the empty site in the macronuclear DNA molecule. These results, in combination with previous studies that have characterized the excised DNA elements, indicate that the two products of excision (the free element and the macronuclear DNA molecule) share DNA sequences. This suggests that excision events are initiated by staggered cuts in the chromosomal DNA.

Animals↗
PubMed1991

Developmentally controlled genomic rearrangements in ciliated protozoa.

The ciliated protozoa undergo an extensive genome reorganization during the course of forming a transcriptionally active macronucleus. The process includes numerous chromosome fragmentation and DNA breakage and rejoining events. Recent work indicates that transposable elements play a role in the process.

Animals↗
PubMed1990

Internal eliminated sequences are removed prior to chromosome fragmentation during development in Euplotes crassus.

The hypotrichous ciliated protozoa undergo a massive genome rearrangement process after their sexual cycle. One frequent type of rearrangement is the removal of DNA sequences (internal eliminated sequences; IESs) from internal regions of DNA molecules. In this study, we characterized the removal of IESs in Euplotes crassus. Southern hybridization analyses combined with cytological observations indicated that IES removal is an early event in macronuclear development, occurring during the polytene chromosome stage and prior to the chromosome fragmentation process. The results are consistent with IES removal occurring via an intramolecular DNA breakage and rejoining process.

Animals↗
PubMed1989

Detection of circular forms of eliminated DNA during macronuclear development in E. crassus.

Following their sexual cycle, hypotrichous ciliated protozoa transform a copy of a chromosomal micronucleus into a macronucleus containing small, linear DNA molecules. A frequent event during macronuclear development is the removal of short segments of DNA (internal eliminated sequences: IESs) by a process equivalent to DNA breakage and rejoining. In this study we used a polymerase chain reaction procedure to demonstrate that free circular forms of IESs are present in cells undergoing macronuclear development. Sequencing of the junctions of the free circular IESs suggests that they share 12 nucleotides with the macronuclear DNA molecules that are generated following IES removal. The results provide evidence that IESs are removed by an active DNA breakage and rejoining process, which may involve staggered cuts in the substrate DNA.

Animals↗
PubMed1989

Characterization of chromosome fragmentation in two protozoans and identification of a candidate fragmentation sequence in Euplotes crassus.

Following the sexual cycle, hypotrichous ciliated protozoans fragment a set of their micronuclear chromosomes to generate the thousands of short, linear DNA molecules present in the transcriptionally active macronucleus. We have used a hybrid selection procedure to examine macronuclear DNA molecules for subtelomeric length heterogeneity to determine whether chromosome fragmentation occurs at unique or multiple sites. The results suggest that multiple, but closely spaced, chromosome fragmentation sites are used by Oxytricha nova. In contrast, Euplotes crassus uses unique chromosome fragmentation sites in a reproducible manner to generate the ends of macronuclear DNA molecules. Additional studies compared DNA sequences in the vicinity of chromosome fragmentation sites in an attempt to define cis-acting sequences that direct the fragmentation process. A conserved sequence was found near chromosome fragmentation sites in E. crassus. The location of the conserved sequence suggests that chromosome fragmentation involves staggered cuts of the micronuclear DNA molecules.

Animals↗
PubMed1989

Micronuclear genome organization in Euplotes crassus: a transposonlike element is removed during macronuclear development.

After mating, hypotrichous ciliated protozoa transform a set of their micronuclear chromosomes into thousands of short, linear DNA molecules that form the macronuclear genome. To examine micronuclear genome organization in the hypotrich Euplotes crassus, we have analyzed two cloned segments of micronuclear DNA as well as the macronuclear DNA molecules that are derived from them. E. crassus was found to display a number of features characteristic of other hypotrich genomes, including (i) clustering and close spacing of the precursors of macronuclear DNA molecules, (ii) the frequent occurrence of internal eliminated sequences within macronuclear precursors, (iii) overlapping macronuclear precursors, (iv) lack of telomeric repeats at the ends of macronuclear precursors, and (v) alternative processing of the micronuclear chromosome to yield multiple macronuclear DNA molecules. In addition, a moderately repetitive, transposonlike element that interrupts the precursors of two macronuclear DNA molecules has been identified and characterized. This transposonlike element, designated Tec1, is shown to be reproducibly removed from one of the macronuclear precursors during independent episodes of macronuclear development.

Animals↗
PubMed1988

Alternative use of chromosome fragmentation sites in the ciliated protozoan Oxytricha nova.

During its life cycle, the hypotrichous ciliated protozoan Oxytricha nova transforms a copy of its micronucleus, which contains chromosome-sized DNA, into a macronucleus containing linear, gene-sized DNA molecules. A region of the micronuclear genome has been defined that gives rise to two distinct macronuclear DNA molecules during development. Through analysis of recombinant macronuclear and micronuclear clones, the generation of the two macronuclear DNA molecules was shown to be the result of alternative use of chromosome fragmentation sites. In addition, evidence was obtained that adjacent micronuclear precursors of macronuclear DNA molecules can overlap by a few base pairs. The significance of these findings in relation to developmental chromosome fragmentation is discussed.

Animals↗
PubMed1988

Genetic characterization and use of a restriction fragment length variant in the hypotrichous ciliate Euplotes crassus.

Two forms of a macronuclear DNA molecule differing in the presence or absence of a restriction endonuclease recognition site have been detected in the hypotrichous ciliate Euplotes crassus. Through a series of genetic crosses the two forms were shown to be allelic, being derived from a single micronuclear genetic locus. This restriction fragment length variant (RFLV) was used as a genetic marker to determine that the migratory and stationary pronuclei generated during mating can be genetically non-identical. In addition, the RFLV was used to investigate the efficiency of processing of the alternate alleles during macronuclear development and their subsequent transmission during vegetative growth. Little or no bias in the processing and/or amplification of the two alleles was observed during macronuclear development. During vegetative growth, however, changes in the relative amounts of the two alleles were observed.

Alleles↗