Genomic organization of mouse Dishevelled genes

We report the isolation and characterization of two new genomic loci corresponding to the mouse Dishevelled (Dvl) genes Dvl2 and Dvl3. The Dvl genes are homologs of the Drosophila dsh segment polarity gene, and are involved in the Wnt/wingless signal transduction pathway. Dvl2 and Dvl3 genomic clones were isolated from a mouse 129 strain λFIXII genomic library and have identical exon/intron organization to Dvll. All three Dishevelled genes span 15 exons and 14 introns and have a number of conserved splice junction sites.     

Characterisation of the beta-tubulin gene of Cylicocyclus nassatus?

mRNA and genomic DNA were isolated from adult Cylicocyclus nassatus, and the mRNA was reverse transcribed. The cDNA was PCR amplified using degenerate primers designed according to the alignment of the β-tubulin amino acid sequences of other species. To complete the coding sequence, the 3′ end was amplified with the 3′-RACE, and for amplification of the 5′ end the SL1-primer was used. The cDNA of the β-tubulin gene of C. nassatus spans 1429 bp and encodes a protein of 448 amino acids. Specific primers were developed from the cDNA sequence to amplify the genomic DNA sequence and to analyse the genomic organisation of the β-tubulin gene. The complete sequence of the genomic DNA of the β-tubulin gene of C. nassatus has a size of 2652 bp and is organised into nine exons and eight introns. The identities with the exons of the gru-1 β-tubulin gene of Haemonchus contortus range between 79% and 97%.     

Purification of triosephosphate isomerase and isolation of its gene from the mosquito Culex tarsalis

The enzyme triosephosphate isomerase (TPI) was purified to homogeneity from the mosquito Culex tarsalis. Anti-C. tarsalis TPI antibodies cross-reacted with TPIs from other organisms but bands on western blots were most intense with proteins from closely related Dipterans. Using a degenerate primer corresponding to the amino-terminal sequence of the protein in a polymerase chain reaction (PCR), a cDNA corresponding to the TPI gene (Tpi) was isolated and sequenced. Subsequently, a genomic sequence including 305 bp to the 5′-end of the coding sequence was obtained. Comparison of C. tarsalis Tpi to that of Drosophila melanogaster revealed that although the two genes had little similarity in the intron and 5′ flanking sequences, they were highly similar (73% identity) in their coding sequence. The rate of synonymous substitution in insect genes may be slower than that of vertebrates, but the nonsynonymous substitution rate, and hence the rate of TPI evolution, appears to be faster in insects than in vertebrates.     

Genomic organization of four β-1,4-endoglucanase genes in plant-parasitic cyst nematodes and its evolutionary implications

The genomic organization of genes encoding β-1,4-endoglucanases (cellulases) from the plant-parasitic cyst nematodes Heterodera glycines and Globodera rostochiensis (HG-eng1, Hg-eng2, GR-eng1, and GR-eng2) was investigated. HG-eng1 and GR-eng1 both contained eight introns and structural domains of 2151 and 2492 bp, respectively. HG-eng2 and GR-eng2 both contained seven introns and structural domains of 2324 and 2388 bp, respectively. No significant similarity in intron sequence or size was observed between HG-eng1 and HG-eng2, whereas the opposite was true between GR-eng1 and GR-eng2. Intron positions among all four cyst nematode cellulase genes were conserved identically in relation to the predicted amino acid sequence. HG-eng1, GR-eng1, and GR-eng2 had several introns demarcated by 5′-GC…AG-3′ in the splice sites, and all four nematode cellulase genes had the polyadenylation and cleavage signal sequence 5′-GAUAAA-3′—both rare occurences in eukaryotic genes. The 5′- flanking regions of each nematode cellulase gene, however, had signature sequences typical of eukaryotic promoter regions, including a TATA box, bHLH-type binding sites, and putative silencer, repressor, and enhancer elements. Database searches and subsequent phylogenetic comparison of the catalytic domain of the nematode cellulases placed the nematode genes in one group, with Family 5, subfamily 2, glycosyl hydrolases from Scotobacteria and Bacilliaceae as the most homologous groups. The overall amino acid sequence identity among the four nematode cellulases was from 71 to 83%, and the amino acid sequence identity to bacterial Family 5 cellulases ranged from 33 to 44%. The eukaryotic organization of the four cyst nematode cellulases suggests that they share a common ancestor, and their strong homology to prokaryotic glycosyl hydrolases may be indicative of an ancient horizontal gene transfer.     

Structural organisation of the rat genes encoding liver- and heart-type of cytochrome c oxidase subunit VIa and a pseudogene related to the COXVIa-L cDNA

To study the tissue-specific expression of the heart(H)- and liver(L)-type of rat cytochrome-c oxidase subunit VIa (rCOXVIa), we have screened and sequenced the genes for the two isoforms. Both genes contain three exons and two introns, spanning 880 bp (rCOXVIa-H) and 3089 bp (rCOXVIa-L), respectively. In both genes, exon I codes for the whole leader sequence comprising 12 (rCOXVIa-H) or 26 (rCOXVIa-L) amino acids and for 12 (rCOXVIa-H) or 10 (rCOXVIa-L) amino acids of the corresponding mature protein, while the remaining amino acids for the mature proteins are encoded by exons II and III. The 5′ region of the genes lack both TATA and CAAT boxes, but show a high G+C content in the early 5′-upstream region. We have identified in upstream regions and in the introns of both genes several putative binding sites associated with respiratory function, muscle gene activation and housekeeping function. In rCOXVIa-H, we identified a CCAC/Myo-D motif, known to be required for muscle-specific expression of the human myoglobin-encoding gene, which is not present in rCOXVIa-L. In addition, we have analyzed a pseudogene, showing 84% homology to the COXVIa-L cDNA sequence.     

Amplification and methylation of an esterase gene associated with insecticide-resistance in greenbugs, Schizaphis graminum (Rondani) (Homoptera: Aphididae)

The greenbug aphid, Schizaphis graminum (Rondani) has developed resistance to organophosphorus insecticides by the over-production of esterases that have been classified as Type I and Type II. The first twenty N-terminal amino acids of the Type I esterase were determined and used to design an oligonucleotide, which in conjunction with an active site primer derived from conserved sequences of other insect esterases and two internal primers specific for esterases from another aphid species resulted in a 0.85 kb genomic DNA fragment from resistant greenbugs. This was extended by 5′ RACE which provided approximately 1.2 kb of the 5′ end of the esterase gene. The 5′ DNA sequence corresponded to 19 of the 20 known amino acids of the Type I esterase, with the last needing only a one base change (probably resulting from a PCR artifact). Furthermore, the sequence showed very close similarity to the amplified E4/FE4 esterase genes of Myzus persicae (Sulzer). A comparison of sequences suggested that the S. graminum gene has introns in the same positions as the first two introns of E4/FE4, with the second intron being considerably larger in S. graminum. Probing of Southern blots with the 0.85 kb esterase fragment showed that the gene encoding the Type I esterase is amplified 4- to 8-fold in resistant S. graminum and that the amplified sequences contain 5-methylcytosine at MspI/HpaII sites, again in agreement with previous findings for M. persicae genes.     

Identification and analysis of a third mouse Polycomb gene, MPc3

Betaine-homocysteine S-methyltransferase (BHMT) is one of the enzymes involved in the branch point metabolism of homocysteine. Elevated levels of plasma homocysteine may be a risk factor for the development of vascular disease; however, whether BHMT has a significant role in the regulation of plasma levels of homocysteine remains to be determined. As a prelude to creating a mouse strain deficient in BHMT activity, we screened a lambda library containing mouse SvJ 129 genomic DNA for the mouse BHMT gene using random probes made from the human cDNA. One genomic isolate was completely sequenced and found to encode an intronless BHMT pseudogene (mBHMT-ps). mBHMT-ps was then used as a template for the generation of random probes that were used to screen a BAC library containing mouse 129 Sv/Ev genomic DNA. In order to discriminate between pseudogenes and the authentic BHMT gene, a secondary PCR-based screen was employed which used primers designed from the pseudogene sequence that would predictably amplify across introns. Using this strategy, we isolated six mouse genomic clones that tested positive for the presence of all seven introns characteristic of the human gene, and the BHMT gene of one clone was completely sequenced. Like the human BHMT gene, the mouse gene spans 21 kb and is encoded by eight exons interrupted by seven introns. The structure of the mouse BHMT gene is described herein as well as the 5′-flanking region of the gene adjacent to exon 1, which we demonstrate is capable of conferring basal promoter activity in Chinese Hamster Ovary cells.     

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 the murine Icam-1 gene.

Intercellular adhesion molecule-1 (ICAM-1, CD54) is a cell adhesion molecule that interacts with the leukocyte beta 2 integrins, LFA-1 and Mac-1. Murine inflammatory models are being utilized increasingly to define the role that ICAM-1 induction plays in the initiation of inflammation. We have isolated murine genomic clones that contain the Icam-1 gene including over 2 kb of 5' flanking sequence. The gene for murine Icam-1 spans over 13 kb and is composed of seven exons and six introns. Each of the extracellular immunoglobulin-like domains of ICAM-1 is encoded by a single exon that ends with the first base of the next codon. Examination of ICAM-1 expression in vivo shows that mRNA levels of ICAM-1 are low in all organs except for the lung but increase markedly in multiple organs at 3 h after administration of endotoxin. The 5' flanking region of the murine gene contains a putative TATA box and potential SP-1, AP-1, and AP-3 sites in positions nearly identical to those in the human gene.