Molecular cloning and nucleotide sequence of a gene encoding a cotton palmitoyl-acyl carrier protein thioesterase.

A cotton genomic clone containing a 17.4-kb DNA segment was found to encompass a palmitoyl-acyl carrier protein (ACP) thioesterase (Fat B1) gene. The gene spans 3.6 kb with six exons and five introns, and is apparently the first plant FatB acyl-ACP thioesterase gene to be completely sequenced. The six exons are identical in nucleotide sequence to the open reading frame of the corresponding cDNA, and would encode a preprotein of 413 amino acids. The preprotein can clearly be identified as a FatB acyl-ACP thioesterase from its similarity to the deduced amino acid sequences of other FatB thioesterase preproteins. A 5'-flanking region of 914 bp was sequenced, with the potential TATA basal promoter 324 bp upstream from the ATG initiation codon. The 5'-flanking sequence also has a putative CAAT box and two presumptive basic region helixloop-helix (bHLH) elements with the consensus motif CANNTG (termed an E box), implicated as being a positive regulatory element in seed-specific gene expression.     

Organization of the mouse microfibril-associated glycoprotein-2 (MAGP-2) gene

A 1.4-kb EST clone encoding mouse microfibril-associated glycoprotein-2 (MAGP-2), identified by its similarity with the reported human cDNA, was used to screen a mouse 129 genomic bacterial artificial chromosome (BAC) library. The mouse gene contains 10 exons spanning 16 kb, located on the distal region of Chromosome (Chr) 6. The exons range in size from 24 to 963 bp, with the ATG located in exon 2. The tenth and largest exon contains 817 bp of 3′ untranslated sequence, including a B2 repetitive element. Northern analysis demonstrates abundant expression of MAGP-2 mRNA in skeletal muscle, lung, and heart. Sequence analysis of additional cDNA clones suggests that the two mRNA forms of MAGP-2 in the mouse arise from alternative polyadenylation site usage. The promoter does not contain an obvious TATA box, and the sequence surrounding the start site does not conform to the consensus for an initiator promoter element. Additionally, the mouse promoter contains 22 copies of a CT dinucleotide repeat sequence located ∼155 bp 5′ to exon 1. Received: 27 August 1999 / Accepted: 2 November 1999     

Structural organization and complete sequence of the human alpha-N-acetylgalactosaminidase gene: homology with the alpha-galactosidase A gene provides evidence for evolution from a common ancestral gene.

Human alpha-N-acetylgalactosaminidase (alpha-GalNAc; EC 3.2.1.49), the lysosomal glycohydrolase that cleaves alpha-N-acetylgalactosaminyl moieties in glycoconjugates, is encoded by a gene localized to chromosome 22q13----qter. The deficient activity of this enzyme results in Schindler disease, an autosomal recessive disorder characterized by the increased urinary excretion of glycopeptides and oligosaccharides containing alpha-N-acetylgalactosaminyl moieties. Recently, the 3.6-kb full-length alpha-GalNAc cDNA sequence was isolated and found to have remarkable nucleotide and predicted amino acid homology (55.8 and 46.9%, respectively) with the human alpha-galactosidase A (alpha-Gal A) cDNA. To investigate the possible evolutionary relatedness of the two glycosidases, the alpha-GalNAc chromosomal gene was isolated and characterized. Screening of a human genomic DNA cosmid library resulted in the identification of a clone, gAGB-1, with an approximately 35-kb insert that contained the entire alpha-GalNAc gene. A single approximately 15-kb EcoRI fragment of gAGB-1, which contained the complete 3.6-kb cDNA sequence, was digested and the subcloned fragments were sequenced in both orientations. The 13,709-bp alpha-GalNAc gene had nine exons ranging from 95 to 2028 bp and intronic sequences of 304 to 2684 bp. All exon/intron junctions conformed to the GT/AG consensus rule. Analysis of 1.4 kb of 5' flanking sequence revealed three Sp1 and two CAAT-like promoter elements. This region was GC-rich (56%), but no HTF island was identified. The gene contained six Alu-repetitive elements, all in the reverse orientation. Comparison of the structural organization of the alpha-GalNAc and the alpha-Gal A genes revealed that all six alpha-Gal A introns were identically positioned in the homologous alpha-GalNAc exonic sequence. Two additional introns, 1 and 8, were identfied in the alpha-GalNAc gene. The predicted amino acid sequences of alpha-GalNAc exons 2 through 7 and those of corresponding alpha-Gal A exons 1 through 6 were 46.2 to 62.7% identical. In contrast, there was little, if any, similarity between the deduced amino acid sequences of alpha-Gal A exon 7 and alpha-GalNAc exons 8 and 9. The remarkable amino acid identity and the identical exonic interruption by six introns of the alpha-GalNAc and alpha-Gal A genes suggest that this region in both genes is evolutionarily related and arose through duplication and divergence from a common ancestral gene.     

Isolation, characterization and expression analysis of a leaf-specific phosphoenolpyruvate carboxylase gene in Oryza sativa.

Suppression subtractive hybridization was carried out to enrich gene fragments over-expressed in rice leaves by subtraction to rice roots, from which two identical cDNA fragments were identified to encode putative phosphoenolpyruvate carboxylase. Then the corresponding full-length cDNA (Osppc) is isolated by RT-PCR and sequenced, which indicates an open reading frame of 2895bp is contained. Its deduced protein is encoded in 10 exons and shows high similarity to many other plant PEPCs. Comparing with maize and bacterial PEPCs, it is revealed that OSPPC shares many conserved domains and active sites that responsible for the structure, activity and regulation of this enzyme. Phylogenetic analysis demonstrates that OSPPC is grouped with C3 form PEPCs of wheat, maize and sorghum, which is consistent with the classification of rice. And a putative promoter element is predicted with DOF binding box, CAAT box and TATA box in the 5'-flanking sequence of Osppc gene. Moreover, Quantitative RT-PCR analyses are performed in hybrid rice and its parents, which show that Osppc is specifically expressed in leaf including leaf vein and sheath.