Nucleotide sequence of the triosephosphate isomerase gene from Aspergillus nidulans: implications for a differential loss of introns

A functional cDNA from Aspergillus nidulans encoding triosephosphate isomerase (TPI) was isolated by its ability to complement a tpi1 mutation in Saccharomyces cerevisiae. This cDNA was used to obtain the corresponding gene, tpiA. Alignment of the cDNA and genomic DNA nucleotide sequences indicated that tpiA contains five introns. The intron positions in the tpiA gene were compared with those in the TPI genes of human, chicken, and maize. One intron is present at an identical position in all four organisms, two other introns are located in similar positions in A. nidulans and maize, and the remaining two introns are unique to A. nidulans. These Aspergillus-specific introns are located in regions of the protein that were predicted to be interrupted by introns based on analysis of a Go plot of chicken TPI. These comparisons are discussed in relation to the evolution of introns within TPI genes.     

Characterisation of a pea hsp70 gene which is both developmentally and stress-regulated

A pea pod cDNA library was screened for sequences specific to lignifying tissue. A cDNA clone (pLP19) encoding the C-terminal region of a hsp70 heat shock protein hybridised only to pod mRNA from pea lines where pod lignification occurred. Expression of pLP19 was induced by heat shock in leaves, stems and roots of pea and chickpea plants. Four different poly(A) addition sites were observed in cDNAs derived from the same gene as pLP19. This gene was fully sequenced; unlike most hsp70 genes, it contains no introns. The 5-flanking sequence contains heat shock elements and other potential regulatory sequences.     

The genomic locus of the human hemopoietic-specific cell protein tyrosine kinase (PTK)-encoding gene (HCK) confirms conservation of exon-intron structure among human PTKs of the src family.

Protein tyrosine kinases (PTKs) are implicated in the control of cell growth by virtue of their frequent appearance as products of retroviral oncogenes, as intracellular signal transducers, and as growth factor receptors or components thereof. The knowledge of the structure and sequence of family genes encoding PTKs is still limited. To date, the complete genomic structure of human src family members is only available for the C-FGR gene (encoding p55 Fgr, PTK). Sequence analysis and characterization of the intron/exon organization of the human HCK gene, encoding a hemopoietic-specific cell PTK of the src-related family, revealed a length of over 16 kb for the seven 3'-exons. All intron/exon splice junctions agree with the GT/AG rule. In each case where a boundary occurs at a Gly codon, GGG or GGA, the triplet is split between the first and second nucleotide (nt). A total of eight complete and one partial Alu repeats were identified within the introns. The nt sequence of the genomic clones resolves existing discrepancies among two published sequences of HCK cDNAs. Human HCK, C-SRC (encoding p60 Src PTK), C-FGR and LCK (encoding p56 Lck, PTK) genes thus share very similar exon/intron structures for the conserved exons. These results provide additional evidence that the different PTKs of the src-like family most likely arose by duplication of an ancestral src-like gene.     

Sequence of EndoA gene encoding mouse cytokeratin and its methylation state in the CpG-rich region.

A genomic clone obtained from mouse liver DNA using a mouse cytokeratin EndoA cDNA probe revealed the complete sequence of the EndoA gene. The gene is divided into nine exons and the exon-intron pattern has been conserved compared to that of other type-II cytokeratin-encoding genes. The 5' upstream, 3' downstream and first and third introns contain potential regulatory sequences, including polyoma virus enhancer motifs (PEA1 and PEA3) and AP-1 elements. The 5' regions upstream of the EndoA, EndoB and Ck8 genes contain homologous sequences surrounding the TATA boxes. In addition, a CpG dinucleotide cluster region was located around the first exon. This CpG cluster region was found to be hypomethylated in endodermal PYS-2 cells, retinoic acid-treated F9 cells, and F9 embryonal carcinoma cells, but hypermethylated in BALB/C 3T3 fibroblast cells that do not express EndoA. These findings may provide a clue to understanding the molecular mechanisms of EndoA gene expression.     

Cloning and characterization of the major histone H2A genes completes the cloning and sequencing of known histone genes of Tetrahymena thermophila.

A truncated cDNA clone encoding Tetrahymena thermophila histone H2A2 was isolated using synthetic degenerate oligonucleotide probes derived from H2A protein sequences of Tetrahymena pyriformis. The cDNA clone was used as a homologous probe to isolate a truncated genomic clone encoding H2A1. The remaining regions of the genes for H2A1 (HTA1) and H2A2 (HTA2) were then isolated using inverse PCR on circularized genomic DNA fragments. These partial clones were assembled into intact HTA1 and HTA2 clones. Nucleotide sequences of the two genes were highly homologous within the coding region but not in the noncoding regions. Comparison of the deduced amino acid sequences with protein sequences of T. pyriformis H2As showed only two and three differences respectively, in a total of 137 amino acids for H2A1, and 132 amino acids for H2A2, indicating the two genes arose before the divergence of these two species. The HTA2 gene contains a TAA triplet within the coding region, encoding a glutamine residue. In contrast with the T. thermophila HHO and HTA3 genes, no introns were identified within the two genes. The 5'- and 3'-ends of the histone H2A mRNAs; were determined by RNase protection and by PCR mapping using RACE and RLM-RACE methods. Both genes encode polyadenylated mRNAs and are highly expressed in vegetatively growing cells but only weakly expressed in starved cultures. With the inclusion of these two genes, T. thermophila is the first organism whose entire complement of known core and linker histones, including replication-dependent and basal variants, has been cloned and sequenced.     

A phylogenetic analysis based on the gene encoding phosphoglycerate kinase

Summary 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.Offprint requests to: R.E. Pearlman     

Complete genomic structure of human rpS3: identification of functional U15b snoRNA in the fifth intron

Analysis of the complete genomic structure of the human ribosomal protein S3 (rpS3) gene revealed the presence of a functional U15b snoRNA gene in its intron. Human ribosomal protein S3 (rpS3) gene of 6115 bp long has been identified to contain six introns and seven exons in this study. The first and fifth introns of human S3 gene contain functional U15 snoRNA genes. Although Xenopus and Fugu counterparts also have six introns and seven exons, S3 gene of Fugu contains two functional U15 snoRNAs in the fourth and sixth introns and two pseudo genes for U15 snoRNAs in the first and fifth introns. In Xenopus S1 gene encoding ribosomal protein S3, however, three of its six introns contain U15 snoRNA gene sequence. Sequence comparison of the U15 genes from Xenopus, Fugu and human revealed that the regions involved in binding to 28S rRNA and the consensus sequence (C, D and D' boxes) for snoRNAs are highly conserved among those genes from these three species. Human U15a and U15b RNAs which are derived from the first and the fifth introns, respectively, have been identified to be functional by microinjection of human U15a and U15b snoRNAs into Xenopus oocyte. Northern blot and primer extension analyses confirm that human U15b snoRNA is expressed in vivo.     

Cloning of the cDNA and Genes for the Hamster and Human β2-Adrenergic Receptors

Abstract

The adenylate cyclase-stimulatory β₂-adrenergic receptor has been purified to apparent homogeneity from hamster lung. Partial amino acid sequence obtained from isolated CNBr peptides was used to clone the gene and cDNA for this receptor. The predicted amino acid sequence for the hamster β₂-adrenergic receptor revealed that the protein consists of a single polypeptide chain of 418 aa with consensus N-glycosylation and phosphorylation sites predicted by previous in vitro data. The most striking feature of the receptor protein however, is that it contains seven stretches of hydrophobic residues similar to the proposed seven transmembrane segments of the light receptor rhodopsin. Significant amino acid homology (30-35%) can be found between the hamster β₂-adrenergic receptor and rhodopsin within these putative membrane spanning regions. Using a hamster β₂-adrenergic receptor probe, the gene and cDNA for the human β₂-adrenergic receptor were isolated, revealing a high degree of homology (87%) between the two proteins from different species. Unlike the genes encoding the family of opsin pigments, of which rhodopsin is a member, the genes encoding both hamster and human β₂-adrenergic receptors are devoid of introns in their coding as well as 5′ and 3′ untranslated nucleotide sequences. The cloning of the genes and the elucidation of the aa sequences for these G-protein coupled receptors should help to determine the structure-function as well as the evolutionary relationship of these proteins.     

Genomic organization of four novel nondisulfide-bridged peptides from scorpion Mesobuthus martensii Karsch: gaining insight into evolutionary mechanism

At least 25 nondisulfide-bridged peptides (NDBPs) have been identified and characterized from scorpions. However, the genomic organization of the genes that encode these peptides have not been reported yet. BmKa1, BmKa2 and BmKb1 are three novel genes that code for NDBPs identified by our group from Mesobuthus martensii Karsch. Based on their cDNA sequences, the genomic DNA sequences encoding these peptides were obtained using the PCR method. Sequence analysis showed that three distinct genomic structural patterns are used to encode these three peptides. The BmKa1 gene is not interrupted by any introns. However, the BmKa2 gene is composed of two exons, interrupted by a 67 bp intron that is located in the DNA region encoding the mature peptide. Two genomic homologues of the BmKb1 cDNA sequence, named BmKb1′ and BmKb2, respectively, were obtained. The BmKb1′ gene contains one intron of 593 bp, inserted into the DNA region that encodes the signal peptide. Similarly, the BmKb2 gene also contains an intron that interrupts the exon that encodes the NDBP signal peptide. The amino acid sequences deduced for BmKb2 and BmKb1′ differ only at one position. The data suggest that the genomic organizational pattern of NDBPs displays more divergence than that exhibited by the genes that encode disulfide-bridged peptides from scorpions.     

Characterization of Urtica dioica agglutinin isolectins and the encoding gene family

Urtica dioica agglutinin (UDA) has previously been found in roots and rhizomes of stinging nettles as a mixture of UDA- isolectins. Protein and cDNA sequencing have shown that mature UDA is composed of two hevein domains and is processed from a precursor protein. The precursor contains a signal peptide, two in-tandem hevein domains, a hinge region and a carboxyl-terminal chitinase domain. Genomic fragments encoding precursors for UDA-isolectins have been amplified by five independent polymerase chain reactions on genomic DNA from stinging nettle ecotype Weerselo. One amplified gene was completely sequenced. As compared to the published cDNA sequence, the genomic sequence contains, besides two basepair substitutions, two introns located at the same positions as in other plant chitinases. By partial sequence analysis of 40 amplified genes, 16 different genes were identified which encode seven putative UDA- isolectins. The deduced amino acid sequences share 78.9–98.9% identity. In extracts of roots and rhizomes of stinging nettle ecotype Weerselo six out of these seven isolectins were detected by mass spectrometry. One of them is an acidic form, which has not been identified before. Our results demonstrate that UDA is encoded by a large gene family.