Evidence for intron capture: an unusual path for the evolution of proteins.
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Animals↗
Biomedical subjects
R E Pearlman
Publications and source records attributed to R E Pearlman.
Evidence for intron capture: an unusual path for the evolution of proteins.
Most new genes are thought to evolve from preexisting genes but duplications of entire genes or shuffling of preexisting exons provides only a limited repertoire of new sequences that can be presented to a cell. Only pieces that previously existed can be used in the construction and any further divergence depends on the slow accumulation of mutations. We show here the presence of a small, in-frame intron in a ciliate phosphoglycerate kinase gene and the insertion of an unusually random amino acid sequence at the same position in trypanosome phosphoglycerate kinase. The unusual sequences in trypanosomes were likely to have originally been introns that have been subsequently captured by the protein and have now been incorporated as part of the coding sequence. Via this path a truly unique sequence can be incorporated into an existing protein, leading in time to the evolution of a new, functionally distinct protein.
Amino Acid Sequence↗
A germ line-specific sequence element in an intron in Tetrahymena thermophila.
In the ciliated protozoa, the germ line micronucleus carries DNA sequences that are eliminated during the developmental program that produces the somatic macronucleus. We have identified such a micronuclear element in an intron of a gene in the holotrichous ciliate, Tetrahymena thermophila. The macronuclear version of this gene, numbered from the transcription initiation site to the polyadenylation site, is 1569 nucleotides long and contains two introns: intron 1 from nucleotides 206 to 382 and intron 2 from nucleotides 665 to 898. The gene is transcribed into a stable polyadenylated mRNA which is predicted to encode a polypeptide with a highly repetitive structure and an unknown function. The micronuclear version of the gene contains a 2.9-kb sequence element, mse2.9, in intron 2. This element was characterized by sequencing two micronuclear genomic clones that contain 909 and 764 base pairs of micronucleus-specific sequence from its left and right ends, respectively. This sequence has an (A+T) content of 81% and is present as a single copy. The termini of mse2.9 are located within a 4-nucleotide direct repeat, TTAT, one copy of which is retained in the macronuclear gene, between positions 755 and 759. Other features near the termini of mse2.9 include a GC-rich sequence motif, CCAACCCGTTG, 31 nucleotides outside the left terminus and a 20-nucleotide palindrome 6 nucleotides outside the right terminus.
Amino Acid Sequence↗
Synaptonemal complex proteins: occurrence, epitope mapping and chromosome disjunction.
We have used polyclonal antibodies against fusion proteins produced from cDNA fragments of a meiotic chromosome core protein, Cor1, and a protein present only in the synapsed portions of the cores, Syn1, to detect the occurrence and the locations of these proteins in rodent meiotic prophase chromosomes. The 234 amino acid Cor1 protein is present in early unpaired cores, in the lateral domains of the synaptonemal complex and in the chromosome cores when they separate at diplotene. A novel observation showed the presence of Cor1 axial to the metaphase I chromosomes and substantial amounts of Cor1 in association with pairs of sister centromeres. The centromere-associated Cor1 protein becomes dissociated from the centromeres at anaphase II and it is not found in mitotic metaphase centromeres. The extended presence of Cor1 suggests that it may have a role in chromosome disjunction by fastening chiasmata at metaphase I and by joining sister kinetochores, which ensures co-segregation at anaphase I. Two-colour immunofluorescence of Cor1 and Syn1 demonstrates that synapsis between homologous cores is initiated at few sites but advances rapidly relative to the establishment of new initiation sites. If the rapid advance of synapsis deters additional initiation sites between pairs of homologues, it may provide a mechanism for positive recombination interference. Immunogold epitope mapping of antibodies to four Syn1 fusion proteins places the amino terminus of Syn1 towards the centre of the synaptonemal complex while the carboxyl terminus extends well into the lateral domain of the synaptonemal complex. The Syn1 fusion proteins have a non-specific DNA binding capacity. Immunogold labelling of Cor1 antigens indicates that the lateral domain of the synaptonemal complex is about twice as wide as the apparent width of lateral elements when stained with electron-dense metal ions. Electron microscopy of shadow-cast surface-spread SCs confirms the greater width of the lateral domain. The implication of these dimensions is that the proteins that comprise the synaptic domain overlap with the protein constituents of the lateral domains of the synaptonemal complex more than was apparent from earlier observations. This arrangement suggests that direct interactions might be expected between some of the synaptonemal complex proteins.
Amino Acid Sequence↗
Common sequence elements are important for transcription and replication of the extrachromosomal rRNA-encoding genes of Tetrahymena.
Three types of conserved sequence elements have previously been identified in the generally non-conserved, 5' non-transcribed spacer region of the rRNA-encoding gene (rDNA) of Tetrahymena species. In vivo and in vitro experiments have identified the start point (tsp) for rDNA transcription. Comparative sequence analysis suggested that the type-I sequence element was likely to function as the promoter for rDNA transcription. We have used in vitro transcription assays to demonstrate that sequences in the 3' half of the type-I repeat located proximal to the tsp are essential for the accurate initiation of rRNA synthesis. These sequences define the 5' boundary of the core or minimal promoter element and include the sequences which function in rDNA replication and maintenance in the homologous regions in two upstream type-I sequence elements. These sequences may bind factors important for both the transcription and replication of rDNA.
Animals↗
A micronucleus-limited sequence family in Tetrahymena thermophila: organization and sequence conservation.
During macronuclear development in the ciliated protozoan Tetrahymena thermophila, sequence reorganization including sequence loss occurs. Addressing questions about the organization and nucleotide sequence of micronucleus limited regions can lead to insights about mechanisms of DNA rearrangements during macronuclear development as well as mechanisms for the maintenance of the stability of micronucleus-limited sequence families. We have previously identified a moderately repetitive micronucleus-limited sequence family called X-H (family members hybridize to an approximately 450 bp Xbal-HindIII restriction fragment), completely absent from macronuclear DNA. The first member of this family which we isolated is associated with terminal sequences characteristic of a Tel-1 element, a putative micronuclear transposable element. Two additional family members have been isolated which are not closely associated with Tel-1 terminal sequences. We have nucleotide sequence data for three cloned members of the X-H family. This analysis has demonstrated that the longest cloned members of the X-H family share a region of homology of approximately 2,400 bp and are highly conserved, differing only by small insertions or deletions of 100 bp or less. The sequences from one of the sequenced family members flanking the region of homology are themselves mostly micronucleus-limited.
Animals↗
Searching for synaptonemal complex proteins and their genes.
As an alternative to the production and use of monoclonal antisynaptonemal complex (SC) antibodies to isolate SC genes, we have explored the use of polyclonal anti-SC antibodies to identify SC genes from a cDNA expression library. The method proved relatively simple, reliable, and fast and has yielded two SC genes. A homologue of one of these genes from a different species has previously been isolated in another laboratory.
Animals↗
A phylogenetic analysis based on the gene encoding phosphoglycerate kinase.
We have determined the nucleotide sequence of both genomic and complementary DNA (cDNA) for the gene encoding the glycolytic enzyme phosphoglycerate kinase from the ciliated protozoan Tetrahymena thermophila. The amino acid sequence for the enzyme has also been derived from the cDNA sequence. The gene contains an open reading frame of 1260 nucleotides encoding 420 amino acids. Coding sequence in genomic DNA is interrupted by two introns at positions corresponding to introns 3 and 4 in mammalian phosphoglycerate kinase genes. The derived amino acid sequence was used to prepare a phylogeny by aligning the Tetrahymena sequence with 25 other phosphoglycerate kinase amino acid sequences. The Tetrahymena sequence is a typical eukaryotic sequence. There is recognizable and clear homology across species that cover nearly the complete range of life forms. The phylogenetic reconstruction of these sequences generally supports the conclusions that have been reached using rRNA sequences.
Amino Acid Sequence↗
Synaptonemal complexes from DNase-treated rat pachytene chromosomes contain (GT)n and LINE/SINE sequences.
Purified chromosome cores (synaptonemal complexes) of rat pachytene chromosomes, from which the chromatin is removed by extensive DNase II digestion, retain a residual class of DNA, presumably the bases of chromatin loops. This synaptonemal complex-associated DNA, isolated by proteinase digestion and phenol extraction of purified DNase-treated synaptonemal complexes, and cloned in plasmid vector pEMBL18, has a length distribution of 50-500 bp. From a library of these fragments, 21 fragments were sequenced. Present in this sample are short 40-200-bp segments with greater than 80% identity to "long" and "short" interspersed repeated elements (LINE/SINEs), an excess of GT/CA tandem repeats and a number of unidentified sequences. The LINE/SINE segments may play a role in homology vs. nonhomology recognition during meiosis and the alternating purine-pyrimidine sequences have been implicated in genetic recombination. Their enrichment in synaptonemal complexes may be related to the synapsis and recombination functions of meiosis.
Animals↗
A phylogenetic analysis based on the gene encoding phosphoglycerate kinase.
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Base Sequence↗
Thymidine kinase from Tetrahymena thermophila. Purification and immunological analysis.
Thymidine kinase is an enzyme involved in DNA precursor metabolism and DNA replication. The synthesis of this enzyme is highly regulated during the cell cycle and the activity of the enzyme is also regulated by feedback inhibition. Genes encoding thymidine kinase have been extremely useful as selectable markers for introducing DNA into a number of cells. In order to study cell cycle regulation of thymidine kinase, the gene which encodes this enzyme, as well as aspects of DNA replication in the ciliated protozoan Tetrahymena thermophila, we have purified thymidine kinase from Tetrahymena. Two forms of thymidine kinase with native molecular masses of 59 kDa and 80 kDa have been identified and purified 6800- and 4600-fold, respectively. The 59-kDa enzyme, a homodimer of 30-kDa subunits, has been purified to near homogeneity and polyclonal antibodies have been raised against the 30-kDa subunit. Serological studies indicate that the two enzymes are antigenically distinct. The antibody against the Tetrahymena protein cross-reacts with a polypeptide in Chinese hamster ovary (CHO) cell extracts of 26 kDa which corresponds to the reported size of Chinese hamster thymidine kinase protein.
Animals↗
Visualization of DNA sequences in meiotic chromosomes.
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Animals↗
Blockage to exonuclease III digestion in the chromatin of Saccharomyces cerevisiae maps to the in vitro--determined binding site of a trans-acting regulatory factor.
A series of transposable element-induced mutations at the HIS4 locus in Saccharomyces cerevisiae have been attributed to the transposition of a Ty element into the 5' regulatory region of this gene. Various Ty-containing His+ revertants have been isolated and the HIS4/Ty junction region sequenced. The only difference found in this region between a His- and a weak His+ strain was a single point mutation, an A----G transition. The position of Ty remained unaltered. Examination of lacZ fusion plasmids further implicated this A----G transition as being responsible for the altered phenotype, the bp transition representing an allele of a cis-acting regulatory element. Subsequent gel retardation and methylation interference experiments revealed that this A----G mutation enabled the binding of a trans-acting factor (TyBf) in vitro. In this paper we show that the TyBf binding site is in a region of chromatin hypersensitive to digestion by DNase I. The binding site is protected in vivo from digestion with exonuclease III, suggesting the presence of a bound protein in His+ ("on") but not His- ("off") Ty-containing strains. We propose that a trans-acting factor binding in vivo, presumably TyBf, is responsible for the activation of HIS4 expression in these insertion mutants.
Binding Sites↗
Telomere and centromere DNA are associated with the cores of meiotic prophase chromosomes.
Mouse (Mus musculus) whole-mount, surface-spread, meiotic prophase chromosomes have an axial core structure, the synaptonemal complex, SC, from which extend chromatin loops. This arrangement permits a novel approach to the analysis of chromosome structure. Using in situ hybridization, the types of DNA sequences preferentially associated with the SC and the types located primarily in the chromatin loops can be determined. With biotinylated probes, detected by avidin conjugated to FITC, we present evidence for differential chromatin-SC interaction. The telomere sequence (TTAGGG)n is associated exclusively with the two ends of each autosomal SC rather than with the chromatin loops. The minor satellite DNA sequences are predominantly localized to the centromeric region of the SC, as defined by CREST serum anti-centromere antibodies. In contrast, the major satellite DNA probe hybridizes to the chromatin loops of the centromeric heterochromatin, and a probe containing a LINE sequence hybridizes to chromatin loops in general with no obvious preference for the SC. These observations demonstrate that, depending on the type of DNA sequence, the chromatin has different properties in regard to its association with the SC.
Animals↗
In situ DNA sequence mapping with surface-spread mouse pachytene chromosomes.
Surface-spread pachytene chromosomes are several times the length of metaphase chromosomes and the decondensed chromatin loops are attached to a well-defined axis (Weith and Traut, 1980). This arrangement permits detailed DNA sequence localization by in situ hybridization. We show that two probes to low-frequency repeated sequences (20 to 50 copies) which locate the centromere proximal in the mouse X metaphase chromosome between bands A1 and A3 (Disteche et al., 1985) and which map 5.5 cM apart (Disteche et al., 1989), hybridize to two distinct chromatin regions 3 to 5 microns apart on a 25 microns long pachytene X chromosome core.
Animals↗
Satellite DNA I in chromatin loops of rat pachytene chromosomes and in spermatids.
Biotinylated rat satellite DNA I probe p93-50 was used to visualize the chromatin of surface-spread rat pachytene chromosomes. Fluorescein isothiocyanate (FITC)-conjugated avidin produces a beaded fluorescence pattern along the chromatin loops that insert in the centromeric region of the synaptonemal complex (SC), the paired cores of homologous chromosomes. The number of fluorescent beads ranges from zero for centromeres without satellite DNA I homologous to probe p93-50, to several hundred for satellite-rich centromeric regions. For the chromosomes that can be identified, the relative amount of satellite DNA is chromosome specific. No satellite DNA I was detected at the non-centromeric ends of the chromosomes or interstitially. DNase-digested nuclei or isolated SCs did not have detectable amounts of satellite DNA in the centromeric regions of the chromosomes or in the residual SCs. The fate of the satellite DNA was followed during spermiogenesis. In the round spermatid the centromeric regions, which appear to be attached to the nuclear envelope, are still distinct and have converging loops of fluorescent chromatin. At later stages there are fewer but still bright fluorescent patches. Satellite DNA I is still detectable in the mature sperm head. These results demonstrate the organization of satellite DNA I in the chromatin loops at the centromeric regions, and they forecast the analysis of chromosome organization in unprecedented detail with a variety of probes in surface spreads of meiotic prophase chromosomes.
Animals↗
Chromatin organization at meiosis.
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Animals↗
Comparison of Tetrahymena ARS sequence function in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe.
We have isolated several Tetrahymena thermophila chromosomal DNA fragments which function as autonomously replicating sequences (ARS) in the heterologous Saccharomyces cerevisiae and Schizosaccharomyces pombe selection systems. The Tetrahymena ARS sequences were first isolated in S. cerevisiae and were derived from non-ribosomal micro- and macronuclear DNA. Sequence analysis of the ARS elements identified either perfect or close matches with the 11 bp S. cerevisiae ARS core consensus sequence. Subcloning studies of two Tetrahymena ARS elements defined functional regions ranging in size from 50 to 300 bp. Testing of the ARS elements in S. pombe revealed that most of the T. thermophila inserts confer ARS function in both yeasts, at least in the sense of promoting a high transformation frequency to plasmids which contain them. However, the actual sequences responsible for ARS activity were not always identical in the two yeasts.
Animals↗