Analyses in transgenic tobacco of the promoter from a Brassica napus gene highly expressed in the stigma

A genomic clone, Pis G363, containing the Brassica napus stigma-expressed gene Pis 63-2 was isolated and sequenced. The coding region of Pis G363 does not possess introns and shows 82% identity to the nucleotide sequence of a gene from Arabidopsis BAC clone T01B08. A 2-kb promoter fragment from Pis G363 was fused to the coding sequence of the marker enzyme β-glucuronidase (GUS) and introduced into tobacco via Agrobacterium-mediated transformation. The promoter fragment directed expression of the GUS gene in the stigma of transgenic tobacco. Some transformants also showed relatively low GUS activity in the pollen. Received: 25 May 1998 / Revision received: 30 July 1998 / Accepted: 21 August 1998     

Expression of constitutive and tissue-specific acyl carrier protein isoforms in Arabidopsis

We have characterized the occurrence and expression of multiple acyl carrier protein (ACP) isoforms in Arabidopsis thaliana (L.) Heynh ecotype Columbia. Immunoblot analysis of ACPs from Arabidopsis tissues separated by native polyacrylamide gel electrophoresis and 1 molar urea polyacrylamide gel electrophoresis revealed a complex pattern of multiple ACP isoforms. All tissues examined (leaves, roots, and seeds) expressed at least three forms of ACP. The immunoblot identifications of ACP bands were confirmed by acylation of ACP extracts with Escherichia coli acyl-ACP synthetase. A full-length cDNA clone has been isolated that has 70% identity with a previously characterized Arabidopsis genomic ACP clone (ACP-1) (MA Post-Beittenmiller, A Hloušek-Radojčić, JB Ohlrogge [1989] Nucleic Acids Res 17: 1777). Based on RNA blot analysis, the cDNA clone represents an ACP that is expressed in leaves, seeds, and roots. In order to identify the protein products of each known ACP gene, their mature coding sequences have been expressed in E. coli. Using polymerase chain reactions, exons II and III of the genomic ACP-1 clone and the mature coding sequences of the ACP-2 cDNA clone were subcloned into E. coli expression vectors. Site-directed mutagenesis was used to convert the amino acid sequence of the ACP-2 cDNA clone to that of the A2 clone of Lamppa and Jacks ([1991] Plant Mol Biol 16: 469-474), ACP-3. The three E. coli-expressed proteins have different mobilities on polyacrylamide gel electrophoresis gels and each comigrates with a different Arabidopsis ACP isoform expressed in leaves, seeds, and roots. Thus, all of the three cloned ACPs appear to be constitutively expressed Arabidopsis ACPs. In addition to these three ACP isoforms, protein blots indicate that seed, leaf, and root each express one or more tissue-specific isoforms.     

Acid phosphatase-1 from nematode resistant tomato : isolation and characterization of its gene

Aps-1 encodes acid phosphatase-1, one of the many acid phosphatases present in tomato (Lycopersicon esculentum Mill.). Aps-1 is closely linked to Mi, a gene conferring resistance against nematodes. Thus, a clone of Aps-1 would provide access to the region of the genome containing Mi. Acid phosphatase-1 was purified from tomato suspension culture cells. Fragmentary amino acid sequences were derived from the purified protein and from its proteolytic and chemical digestion products. One of these amino acid sequences was used to design an oligodeoxyribonucleotide probe expected to hybridize to acid phosphatase-1 cDNA. This probe identified, in a cDNA library, a clone encoding the carboxyl-terminal sequence of a protein that is very similar, but not identical, to acid phosphatase-1. Using this clone, we discovered a second cDNA clone that corresponds in its carboxyl terminal sequence to acid phosphatase-1 but, surprisingly, retains sequences of an Aps-1 intron. The second cDNA clone was used to detect both a cDNA clone and a genomic clone corresponding to Aps-1. The identity of these clones was confirmed by sequence analysis and by the correlation of a restriction fragment length polymorphism with two Aps-1 alleles in a segregating tomato population. The deduced amino acid sequence of the Aps-1 open reading frame predicts a hydrophobic animoterminal signal sequence and a mature protein with a molecular weight of 25,000. The amino acid sequence of this protein has a strong similarity in size and sequence to a vegetative storage protein of soybean.     

Structure and Expression of a Heat-Shock Protein 83 Gene of Pharbitis nil

Four cDNA clones representing mRNAs whose levels were affected by a photoperiod that induces flowering in Pharbitis nil were isolated by a differential hybridization screening procedure. The level of mRNAs represented by three clones (12L, 15L, and 17L) increased following a photoperiod that induces flowering and that represented by the fourth clone (clone 27) increased under conditions in which flowering was inhibited. DNA sequence analysis showed that one cDNA, clone 17L, is homologous to members of the 83- to 90-kD heat-shock protein (hsp) gene family. The corresponding gene, hsp83A, was isolated and its DNA sequence was determined. hsp83A encodes a protein that exhibits 70% amino acid identity with Drosophila melanogaster HSP83. The P. nil hsp83A gene contains two introns within the coding region. hsp83A mRNA was not detectable in cotyledons of plants grown in continuous light, but its level increased transiently following a 14-h dark period and reached a maximum 2 h after the lights were turned on. A dramatic increase in the level of hsp83A mRNA was also found 2 h after an end-of-day dark treatment. Genomic Southern blot analysis demonstrated that the P. nil hsp83-90 gene family consists of at least six members, one of which appears to be constitutively expressed in the light.     

Identification, cloning, and expression of a GHF9 cellulase from Tribolium castaneum (Coleoptera: Tenebrionidae)

The availability of sequenced insect genomes has allowed for discovery and functional characterization of novel genes and proteins. We report use of the Tribolium castaneum (Herbst) (red flour beetle) genome to identify, clone, express, and characterize a novel endo-β-1,4-glucanase we named TcEG1 (T. castaneum endoglucanase 1). Sequence analysis of a full-length TcEG1 cDNA clone (1356 bp) revealed sequence homology to enzymes in glycosyl hydrolase family 9 (GHF9), and verified presence of a change (Gly for Ser) in the conserved catalytic domain for GHF9 cellulases. This TcEG1 cDNA clone was predicted to encode a 49.5 kDa protein with a calculated pI of 5.39. Heterologous expression of TcEG1 in Drosophila S2 cell cultures resulted in secretion of a 51-kDa protein, as determined by Western blotting. The expressed protein was used to characterize TcEG1 enzymatic activity against two cellulose substrates to determine its specificity and stability. Our data support that TcEG1 as a novel endo-β-1,4-glucanase, the first functional characterization of a cellulase enzyme derived from an insect genome with potential applications in the biofuel industry due to its high relative activity at alkaline pH.     

Cloning, sequencing and expression of cDNA encoding growth hormone from Indian catfish (Heteropneustes fossilis)

A tissue-specific cDNA library was constructed using polyA⁺ RNA from pituitary glands of the Indian catfishHeteropneustes fossilis (Bloch) and a cDNA clone encoding growth hormone (GH) was isolated. Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3′ region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length cDNA clone is 1132 bp in length, coding for an open reading frame (ORF) of 603 bp; the reading frame encodes a putative polypeptide of 200 amino acids including the signal sequence of 22 amino acids. The 5′ and 3′ untranslated regions of the cDNA are 58 bp and 456 bp long, respectively. The predicted amino acid sequence ofH. fossils GH shared 98% homology with other catfishes. Mature GH protein was efficiently expressed in bacterial and zebrafish systems using appropriate expression vectors. The successful expression of the cloned GH cDNA of catfish confirms the functional viability of the clone.     

An early meiosis cDNA clone from wheat

Meiosis occupies only a very short period of the life cycle of higher plants but it is a crucial process ensuring the correct passage and maintenance of genetic information from parent to offspring. A clone (designated pAWJL3) has been isolated from a cDNA library generated from RNA prepared from young wheat florets at early meiosis. The clone was identified through cross-hybridisation to a cDNA clone from maize that, in turn, had been isolated by hybridisation to a Lilium meiosis-specific cDNA clone. The genes encoding the sequence represented in the wheat cDNA clone have been assigned to chromosomes in wheat. The clone, pAWJL3, represents a small family of genes with about 20 members located on the short arms of group 3 and 5 chromosomes. The chromosomal regions harbour genes known to control chromosomal pairing in wheat. DNA prepared from a deletion mutation affecting one of the major genes controlling pairing, Ph2 located on the short arm of 3DS, lacks the 3DS-specific members of the pAWJL3 family bands. The genes are shown to be expressed only after leptotene and predominantly at zygotene and pachytene of meiosis I. The deduced amino acid sequence encoded by the cDNA clone shows two domains, one with three leucine-rich, 24-amino acid repeats and the other with four leucine heptad repeats that resemble those found in basic leucine zipper proteins.     

A naturally occurring 46-amino acid deletion of cytidine monophospho-N-acetylneuraminic acid hydroxylase leads to a change in the intracellular distribution of the protein

Cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) hydroxylase is a key enzyme for the expression ofN-glycolylneuraminic acid. The molecular cloning of this enzyme from mouse liver has been described in our previous report (Kawano T, Koyama S, Takematsu H, Kozutsumi Y, Kawasaki H, Kawashima S, Kawasaki T, Suzuki A (1995)J Biol Chem 270: 16458–63). During the cDNA cloning, a cDNA containing a truncated open reading frame (ORF) was isolated. This clone encodes a protein of 531 amino acids which lacks 46 amino acids in the middle of the normal full-length protein. The percentage of this mRNA containing the truncated ORF out of the total population of CMP-NeuAc hydroxylase mRNA in various mouse tissues was about 10–25%. The truncated protein was expressed in COS-1 cells, but did not show any enzymatic activity. The truncated protein was localized to the region which appeared to be the endoplasmic reticulum, whereas the full-length protein with normal enzymatic activity was detected in the cytosol. These data suggest that this naturally occurring 46-amino acid deletion leads to a change in the intracellular distribution of CMP-NeuAc hydroxylase, and a loss in the activity of this enzyme.