trans activation of the Escherichia coliato structural genes by a regulatory protein from Bacillus megaterium : potential use in polyhydroxyalkanoate production

A Bacillus megaterium genomic fragment, which encoded an activator homologous to σ⁵⁴ regulators and which was capable of activating Escherichia coli ato genes in trans, was detected in a gene library of B.␣megaterium screened for β-ketothiolase activity. The fragment presented only one complete open reading frame (ORF1), which encoded a protein of 398 amino acids. The recombinant plasmid complemented mutations in the Escherichia coli atoC regulatory gene. The constitutive expression of the E. coli ato operon mediated by ORF1 could be useful for the synthesis of polyhydroxyalkanoates with different flexibility properties by recombinant E. coli strains. Received: 20 October 1997 / Received revision: 18 February 1998 / Accepted: 23 February 1998     

Molecular cloning and characterization of <Emphasis Type="Italic">GhAPm</Emphasis>, a gene encoding the μ subunit of the clathrin-associated adaptor protein complex that is associated with cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>) fiber development

The clathrin-associated adaptor protein (AP) complexes are the primary clathrin adaptors that contribute to the formation of clathrin-coated vesicles (CCVs). The GhAPm gene (GenBank accession number: GU359054), which encodes the medium subunit of the AP complexes, was cloned from cotton by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The full-length cDNA was 1590 bp in size and encoded an open reading frame (ORF) of 416 amino acids with a molecular weight of 46 kDa. The GhAPm protein shared 81–85% identity at the amino acid level with the AP complex μ subunits isolated from Vitis vinifera, Glycine max, Populus trichocarpa, Ricinus communis and Arabidopsis thaliana, respectively. The corresponding genomic DNA, containing eight exons and seven introns, was isolated and analyzed. Also, a 5′-flanking region was analyzed, and a group of putative cis-acting elements were identified. DNA gel blot analysis showed that there is only one GhAPm gene in the cotton genome. Real-time RT-PCR analysis revealed that GhAPm is expressed in the root, stem, leaf, petal, ovule, and fiber. However, the interesting finding is that GhAPm expression level was shown to increase steadily as the cotton fiber develops. In 30 DPA fibers, expression increases sharply and arrives at a peak then the expression levels decrease rapidly. Based on these data, we propose that GhAPm has a critical role in cotton membrane trafficking and fiber development.     

Molecular cloning and characterization of an inducible RNA-dependent RNA polymerase gene, <Emphasis Type="Italic">GhRdRP</Emphasis>, from cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.)

The RNA-dependent RNA polymerase (RdRP) cDNA, designated as Gossypium hirsutum RdRP (GhRdRP) was cloned from cotton by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The full-length cDNA was 3,672 bp in size and encoded an open reading frame (ORF) of 1,110 amino acids which contained the RdRP conserved functional domain and the signature motif DbDGD. Amino acid sequence alignment indicated that GhRdRP shared the highest identity (66.37%) with AtRdRP1 and had homology with other plant, fungal, yeast and nematode RdRPs. The corresponding genomic DNA containing five exons and four introns, was isolated and analyzed. Also a 5′-flanking region was cloned, and a group of putative cis-acting elements were identified. Southern blot analysis revealed a single copy of the GhRdRP gene in cotton genome. The expression analysis by semi-quantitative RT-PCR showed that GhRdRP was induced by salicylic acid (SA), 5-chloroSA (5-CSA) and fungal infection of Rhizoctonia solani Kuhn. The cloning and characterization of the GhRdRP gene will be useful for further studies of biological roles of GhRdRP in plants.     

Cloning and bioinformatics analysis of a novel acidophilic β-mannanase gene,Auman5A,from Aspergillus usamii YL-01-78

The full-length cDNA sequence, which encodes a novel acidophilic β-mannanase (abbreviated as AuMan5A) of Aspergillus usamii YL-01-78, was amplified by 3′ and 5′ rapid amplification of cDNA ends (RACE) using the total RNA as template. The cDNA sequence is 1,427 bp in length, including 5′ and 3′ non-coding regions and an open reading frame (ORF). The ORF encodes a 21-aa signal peptide, a 17-aa propeptide, and a 345-aa mature peptide (AuMan5A) with the calculated M.W. of 37,614 Da and pI of 4.09 and two putative N-glycosylation sites. Online analysis of amino acid sequence homology demonstrated that the AuMan5A belongs to the glycoside hydrolase (GH) family 5. Its three-dimensional structure was predicted using Pred3D Web Server 1.0 based on the crystal structure of the T. reesei RutC-30 β-mannanase (1QNO) from the GH family 5. Furthermore, the complete DNA sequence encoding the AuMan5A, designated as Auman5A, was cloned from the genomic DNA of A. usamii YL-01-78 by the conventional PCR and pUCm-T vector-mediated PCR techniques. The cloned Auman5A is 2,168 bp in length, harboring 5′ and 3′ flanking regulatory regions and the full-length cDNA sequence in which two short introns with 63 and 60 bp are inserted, respectively.     

Molecular cloning and analysis of a cDNA coding for the bifunctional dihydrofolate reductase-thymidylate synthase of Daucus carota

Molecular cloning of dihydrofolate reductase-thymidylate synthase (DHFR-TS) of Daucus carota was achieved by immunoscreening of a cDNA library obtaining a 2 kbp clone which contains an open reading frame of 1528 bp. Comparison of the deduced amino acid sequence with those from other sources revealed the presence of motifs typical of DHFR and TS thus confirming the bifunctional nature of the carrot protein. As in other organisms, a higher degree of conservation was observed in the TS domain. Analysis of the dhfr-ts gene content in carrot revealed the presence of several copies per diploid genome.     

A large open reading frame ( orf1995 ) in the chloroplast DNA of Chlamydomonas reinhardtii encodes an essential protein

An open reading frame potentially encoding a protein of 1995 amino acids (orf1995) has been found in the chloroplast genome of the green alga Chlamydomonas reinhardtii. Besides having a short hydrophobic N-terminal domain with five putative transmembrane helices, the predicted orf1995 product is highly basic. orf1995 might be a homologue of the ycf1 gene in land plants, whose function has not yet been determined. Mutants of C. reinhardtii transformed with a disruption of orf1995 remain heteroplasmic for the wild-type and disrupted alleles of this gene, indicating that the orf1995 product is essential for cell survival. Received: 18 August 1996 / Accepted: 24 September 1996     

Molecular cloning and characterization of caprine <Emphasis Type="Italic">calpastatin</Emphasis> gene

Calpastatin (CAST) is an important gene for meat quality traits in livestock and poultry. The cDNA of caprine CAST gene was amplified for the first time using RACE-PCR. Results showed the full-length cDNA of caprine CAST gene (Accession no. GU944861) was 2435 base pair (bp) and contained a 2187 bp open reading frame encoding a protein with 728 amino acid residues. Bioinformatic analysis indicated that caprine CAST cDNA was 89.8–95.4, 83.5–92.2, 72.8–81.8 and 69.8–73.5% identical to sheep, cattle, pig and human CAST cDNA. It was predicted that caprine CAST contained four conserved domains with 42 serine phosphorylation loci, 18 threonine phosphorylation loci, 1 tyrosine phosphorylation locus and 5 specific PKC phosphorylation loci. This work provided an important experimental basis for further research on the function of CAST in goat.     

Cloning and characterization of the gene encoding a repressible acid phosphatase (PHO1) from the methylotrophic yeast Hansenula polymorpha

A cloned cDNA, generated from mRNA isolates of phosphate-derepressed H. polymorpha cells, was identified to harbour an incomplete sequence of the coding region for a repressible acid phosphatase. The cDNA fragment served as a probe to screen a plasmid library of H. polymorpha genomic DNA. A particular clone, p606, of a 1.9-kb insert contained a complete copy of the PHO1 gene. Sequencing revealed the presence of a 1329-nucleotide open reading frame encoding a protein of 442 amino acids with a calculated M _r of 49400. The␣encoded protein has an N-terminal 17-amino-acid secretory leader sequence and seven potential N-glycosylation sites. The leader cleavage site was confirmed by N-terminal sequencing of the purified enzyme. The nucleotide sequence is 48.9% homologous, the derived amino acid sequence 36% homologous to its Saccharomyces cerevisiae counterpart. The derived amino acid sequence harbours a consensus sequence RHGXRXP, previously identified as a sequence involved in active-site formation of acid phosphatases. The PHO1 promoter and the secretion leader sequence present promising new tools for heterologous gene expression. Received: 15 January 1998 / Received revision: 2 March 1998 / Accepted: 4 March 1998     

Cloning and Sequencing of Cytochrome P450 1A Complementary DNA in Eel (Anguilla japonica)

Cytochrome P450 1A (CYP1A) complementary DNA was isolated from eel (Anguilla japonica) liver treated with 3-methylcholanthrene. The cDNA contained a 5′ untranslated region of 163 bp, an open reading flame of 1560 bp coding for 519 amino acids and a stop codon, and a 3′ untranslated region of 1730 bp. The predicted molecular weight was approximately 58.4 kDa. The deduced amino acid sequence exhibited identities with reported CYP1A sequences of 80% for rainbow trout, 79% for scup, 76% for plaice and butterfly fish, and 74% for toadfish. When compared with mammalian CYP proteins, the eel CYP1A was more similar to CYP1A1 (54%–56%) than to CYP1A2 (49%–52%). Northern and Southern blot analyses showed two distinct bands, suggesting the existence of another 3-methylcholanthrene-inducible CYP1A gene in eel. Received December 19, 1998; accepted February 18, 1999     

Abundant mRNAs specific to the developing cotton fibre

 Five fibre-specific cDNA clones were isolated by differential screening of a cDNA library from cotton fibres, a developmentally synchronous population of non-dividing cells. The genes corresponding to these cDNAs were expressed preferentially in fibre cells and exhibited differing patterns of temporal expression during fibre development. One cDNA encoded a lipid transfer protein (LTP), and a second encoded a member of a group of well-characterised proline-rich proteins (PRP) from plants. The presence of signal peptide-encoding sequences suggests that both the LTP and the PRP are targeted to the extracellular matrix of the fibre, and a role is envisaged for each in cell elongation. Sequence analysis showed that a third clone was similar to a previously reported fibre-specific sequence of unknown function, whilst the remaining two cDNA clones showed no sequence similarity to previously reported plant nucleic acids. Received: 24 September 1996 / Accepted: 18 October 1996