Genetic engineering in the Precambrian: structure of the chicken triosephosphate isomerase gene.

Abstract

We report the sequence of the single chicken triosephosphate isomerase gene and its flanking regions. The 3-kilobase-long gene is composed of seven similarly sized exons and six introns. By using crystallographic and sequence data, we argue that this ancient gene was originally assembled from the genetic antecedents of exons.Although eucaryotic genes split by introns are now accepted as commonplace, the origin of introns remains enigmatic. Gilbert (24,25) proposed that exons are the descendants of ancient genes encoding small polypeptides with simple functions that were duplicated and reassorted to create genes for more complex proteins. This hypothesis was supported by the fact that the exons of the immunoglobulin genes corresponded to the functional and structural domains of antibody proteins (10,20,42,49). The correlation is also apparent for collagen (50, 52), ovomucoid (46), a-fetoprotein (21), and -y-crystallin (37), which are divided in a way that reflects the repeating nature of the proteins that they encode. Blake (7,8) Southern hybridization. Genomic chicken DNA (embryonic muscle; 20 ,ug per lane) was separated on an agarose gel (1%), blotted to nitrocellulose, and hybridized with the cDNA probe (44, 51).Sequencing. The 9.3-kilobase fragment of Charon 4a bacteriophage 4)3, which contains the entire TIM gene, was cloned in both orientations into the PstI site of pBR322 (48). A series of nested deletions of the resultant plasmids, pgt7 and pgt9, was generated by a modification of the method of Lehrach (22). Plasmid DNA (100 ,ug) that had been linearized by DNase I digestion (500 pg DNase I in 0.5 ml for 20 min at 25°C) was made blunt ended by treatment with BAL 31 nuclease (1 U in 0.04 ml for 1 min at 37°C). After digestion with EcoRI, the ends were filled in with DNA polymerase (Klenow fragment). Nested deletions staggered by about 250 base pairs (bp) were obtained in either of two ways. (i) DNA, treated as described above, was sized on a 1% low-melting agarose gel (SeaKem; FMC Corp., Marine Colloids Div., Rockland, Maine). Gel slices were extracted with phenol, and the DNA was used to transform portions of frozen competent Escherichia coli HB101 (9, 16). The largest-sized fractions were digested with PvuI before transformation to prohibit transformation by full-length plasmids which had escaped DNAase I digestion. (ii) DNA was directly ligated and used to transform strain HB101. Supercoiled plasmid DNA from mini-preps was sized on agarose gels with the aid of supercoiled markers (gift of W. Herr and V. Corbin).After cutting with ClaI and labeling with [a32P]dCTP (>5,000 Ci/mmole; New England Nuclear) by DNA polymerase (Klenow fragment), plasmids were sequenced by the method of Maxam and Gilbert (36). Both strands of the DNA 3497 on May 11, 2018 by guest

Intron positions correlate with module boundaries in ancient proteins

“…Is there a statistically significant excess of intron positions within the linker regions? We first examine 28-Å modules, since modules of this size were defined by Mitiko Go for her analysis of hemoglobin and her prediction of a novel intron (12) and were used again for the prediction of introns in triosephosphate isomerase (13,14). Table 2 shows The last column lists the PDB entry that yielded the coordinates.…”

“…Although a variety of arguments for early introns have been advanced, some of which include the use of the module hypothesis to predict the existence of certain introns (12)(13)(14) while others involve the coincidence of intron positions in genes separated by great evolutionary distances (13), arguments for the introns-late view have recently appeared.…”

Intron‐dependent evolution: Preferred types of exons and introns

“…In addition to the cases listed above, exon shuffling has been claimed to have played a role in the formation of the genes of phosphoglycerate kinase [64], alcohol dehydrogenase [65-681, pyruvate kinase [69], glyceraldehyde-3-phosphate dehydrogenase [70,71], triosephosphate isomerase [72,73], dihydrofolate reductase and serine protease catalytic chains [74]. The present intron pattern of these genes, however, does not show the regularities in intron phase usage expected of genes that were assembled by exon shuffling.…”

Triose‐phosphate isomerase of Leishmania mexicana mexicana Cloning and characterization of the gene, overexpression in Escherichia coli and analysis of the protein

“…The 32 residues constituting the inferface of 7: brucei are underlined [8,91. Sequences presented are from: 7: brucei [13], human [28], chicken [29], yeast [30] and E. coli [31].…”