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.     

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.     

Fruit ripening-related expression of a gene encoding group 5 late embryogenesis abundant protein inCitrus

A cDNA and genomic clone (CuLEA5) encoding a group 5 late embryogenesis abundant protein (Lea5) was isolated from citrus fruit cDNA and genomic libraries. Sequence analysis indicated that the clone contains an open reading frame of 97 amino acids, and that the genomic structure is composed of two exons and one intron. A comparison of its amino sequence with other plant proteins showed that Lea5 proteins can be classified into two types - gymnosperm and angiosperm — based on a P-segment sequence designated by this study. Examination of its expression patterns indicated thatCuLEA5 has important roles during the development or ripening of seedless fruits and leaves inCitrus. The 5′-flanking region of the genomic DNA contains a number of putative hormonal- and stress-responsive elements. This is the first report that describes the expression ofLea5 during fruit ripening, as well as the sequence characteristics of its promoter region.     

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.     

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.     

Evolution of cis-Acting Elements in 5′ Flanking Regions of Vertebrate Actin Genes

Regulation of the vertebrate actin multigene family involves the recognition of various regulatory sequences (cis-acting elements) that specify the distinct tissue type and developmental program of expression for each actin paralogue, which implies that the distribution of cis-acting elements may be unique for each paralogue gene. To elucidate the evolution of these unique distribution patterns, we improved a method to scan for cis-acting elements in the 5′ flanking regulatory region of genes and used it to analyze five cis-acting elements (SRE, MyoD binding site, Elk-1 binding site, positive and negative YY1 binding sites) of six actin paralogue genes (β and γ cytoplasmic actins, α and γ smooth muscle actins, and α skeletal and α cardiac actins) among various vertebrates. It was shown that although an element(s) may exist in all paralogue genes of the same species, its numbers, compositions, and distribution patterns or even sequences vary remarkably among paralogues, which contributes to their different tissue- and developmental-specific expression. However, each pair of coexpressed paralogues has some certain similarity in distribution patterns. Furthermore, among various orthologues of actin genes derived from diverse vertebrates, the sequences, numbers, and distribution patterns of these cis-acting elements are highly conserved or even identical in the long run of phylogeny of vertebrates. Taken together, the results described above strongly indicate that not only the structures of actins but also their expression patterns are essential in both the phylogeny and the physiology of vertebrates. The distribution patterns of cis-acting elements of various actin genes can be regarded as indicators of both horizontal (paralogous) and vertical (orthologous) evolution of actins. Received: 1 March 1999 / Accepted: 6 August 1999     

Sequence analysis of two tandemly linked Em genes from wheat

DNA sequences are presented for two members of the wheat Em gene family. The sequences correspond to the two linked genes at the Xem-1AL locus. Comparisons of these sequences with that of another wheat Em gene and two Em cDNA clones reveals substantial homology within the protein-coding regions, and the presence in the 5-flanking regions of the genomic sequences of motifs characteristic of ABA-responsive cis-acting elements.     

The Tall Fescue Turf Grass Class I Chitinase Gene <Emphasis Type="Italic">FaChit1</Emphasis> Is Activated by Fungal Elicitors,Dehydration, Ethylene,and Mechanical Wounding

The cDNA, genomic DNA, and promoter sequence of FaChit1, a class I chitinase gene from Festuca arundinacea, were isolated and characterized in the present work. The deduced amino acid sequence of FaChit1 contains the chitin binding, catalytic, and proline and glycine-rich domains characteristic for most class I chitinases, but no C-terminal extension region. FaChit1 is induced effectively by fungal elicitors, dehydration, and ethylene, but only slightly by mechanical wounding. To identify potential stress-related cis-acting elements, 5′ sequences 935, 651, and 233 bp upstream of the FaChit1 start codon were fused to the GUS reporter gene and analyzed in transgenic tobacco. The results indicated that the 935 bp fragment closely mirrored endogenous gene expression and that the 651 bp fragment was sufficient to direct reporter the gene expression in response to fungal elicitors, ethylene, dehydration, or mechanical wounding due to both known and presently uncharacterized cis-acting elements. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genomic Cloning and Characterization of a Novel Lectin Gene from <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis>

A new lectin gene was isolated by using genomic walker technology and revealed to encode a mannose-binding lectin. Analysis of a 2233 bp segment revealed a gene including a 1169 bp 5′ flanking region, a 417 bp open reading frame (ORF) and a 649 bp 3′ flanking region. There are two putative TATA boxes and eight possible CAAT boxes lie in the 5′ flanking region. The ORF encodes a 15.1 kDa precursor, which contains a 24-amino acid signal peptide. One possible polyadenylation signal is found in the 3′-flanking region. No intron was detected within the region of genomic sequence corresponding to zaa (Zantedeschia aethiopica agglutinin) full-length cDNA, which is typical of other mannose-binding lectin gene that have been reported. The deduced amino acid sequence of the lectin gene coding region shares 49–54% homology with other known lectins. The cloning of this new lectin gene will allow us to further study its structure, expression and regulation mechanisms.     

Cloning and characterization of a gene encoding wheat starch synthase I

 A cDNA clone, and a corresponding genomic DNA clone, containing full-length sequences encoding wheat starch synthase I, were isolated from a cDNA library of hexaploid wheat (Triticum aestivum) and a genomic DNA library of Triticum tauschii, respectively. The entire sequence of the starch synthase-I cDNA (wSSI-cDNA) is 2591 bp, and it encodes a polypeptide of 647 amino-acid residues that shows 81% and 61% identity to the amino-acid sequences of SSI-type starch synthases from rice and potato, respectively. In addition, the putative N-terminal amino-acid sequence of the encoded protein is identical to that determined for the N-terminal region of the 75-kDa starch synthase present in the starch granule of hexaploid wheat. Two prominent starch synthase activities were demonstrated to be present in the soluble fraction of wheat endosperm by activity staining of the non-denaturing PAGE gels. The most anodal band (wheat SSI) shows the highest staining intensity and results from the activity of a 75-kDa protein. The wheat SSI mRNA is expressed in the endosperm during the early to mid stages of wheat grain development but was not detected by Northern blotting in other tissues from the wheat plant. The gene encoding the wheat SSI (SsI-D1) consists of 15 exons and 14 introns, similar to the structure of the rice starch synthase-I gene. While the exons of wheat and rice are virtually identical in length, the wheat SsI-D1 gene has longer sequences in introns 1, 2, 4 and 10, and shorter sequences in introns 6, 11 and 14, than the corresponding rice gene. Received: 5 June 1998 / Accepted: 29 September 1998     

Characterization of the rat carbonic anhydrase II gene structure: sequence analysis of the 5′ flanking region and 3′ UTR

The rat carbonic anhydrase II gene was characterized and found to be approximately 15.5 kb in length and to contain 7 exons and 6 introns. All intron/exon junction and branch point sequences conform to consensus sequences, and the overall rat CA II genomic structure appears to be conserved upon comparison with mouse, human, and chicken CA II genes. The putative cis-acting elements within the analyzed 1014 bp 5′ flanking region include: TATA box, 4 Sp1 binding sites, 2 AP2 sites and putative tissue-specific β-globin-like repeat elements. A CpG island of approximately 800 bp was identified that begins about 600 bp upstream of exon 1 and extends about 200 bp into intron 1. In the 3′ UTR, two polyadenylation signals (AATAAA) are present, the second of which is believed to be utilized. Northern blot analysis reveals that the 1.7 kb rat CA II mRNA is abundantly expressed in adult male brain and kidney, while negligible amounts are detected in heart and liver.     

Molecular cloning and structural analysis of the cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) phosphoenolpyruvate carboxykinase (<Emphasis Type="Italic">CsPCK</Emphasis>) gene

Genomic sequence of the ATP-dependent phosphoeno/pyruvate carboxykinase (CsPCK) gene has been determined first from cucumber. Several putative clones were isolated in three rounds of genomic library screening with designated cDNA probes. These clones were analyzed via restriction digests, Southern hybridization, and nucleotide sequencing to ascertain the structure of theCsPCK gene. Analysis of a selected positive clone (λcscpk-4A) demonstrated that this gene consists of 13 exons and 12 introns, spanning 9 kb in the cucumber genome. Exon 1 contains only 23 nucleotides of the 5′-noncoding region of cucumberPCK cDNA, whereas Exon 2 comprises 12 nucleotides of the S′-noncoding region with an N-terminal PEPCK coding sequence. All the exon-intron junction sequences agree with the GT/AG consensus, except for the 5 donor site of Intron 7, where GC replaces the GT consensus. As with rice (Oryza sativa), cucumber contains only one copy of theCsPCK gene in its haploid genome. The overall number of exons and the structure of this gene are similar to those for bothArabidopsis Chromosome 4 (Atg4)PCK and the rice PCX genes, which contain 13 and 12 exons, respectively. Two additionalArabidopsis PCK genes can be found in the fifth chromosome (Atg5), which contains 9 exons and 8 introns (with 628 and 670 amino acids, respectively) of the PEPCK peptide. TheCsPCK gene promoter has conserved plant-specific as-acting elements within 2 kb of the 5’ flanking region. Several common cis-acting elements of the isocitrate lyase (icl) and malate synthase(ms) gene promoters, identified in theCsPCK gene, are responsible for the sugar response during plant development, especially at germination. These conserved elements are discussed here.     

Isolation and characterization of a harvest-inducible gene <Emphasis Type="Italic">hi11</Emphasis> and its promoter from alfalfa

The harvesting and storing of alfalfa is a routine practice in the agricultural industry worldwide. To investigate gene expression in harvested alfalfa, cDNA from non-harvested and harvested plants in the field was subjected to subtractive hybridization to identify, in particular, those genes that are induced by the harvesting treatment. One cDNA clone, named hi11, was isolated and analysed. The full length cDNA of the hi11 gene was cloned by RACE amplification. The hi11 gene, which has high homology to a putative protein of unknown function in Arabidopsis, was induced in alfalfa following harvesting, a 38°C heat shock and a wounding treatment. Northern blot analysis confirmed that the expression patterns of hi11 in alfalfa in response to harvesting, heat shock, and wounding. In addition, genomic walking was performed to isolate the 5′ flanking region of the hi11 gene. The promoter of the hi11 gene was fused to the GUS reporter gene and transferred to Medicago truncatula and tobacco. In all transgenic plants of M. truncatula and tobacco, GUS gene expression was observed in harvested tissue, especially in the transgenic tobacco plants, but not in the non-harvested control tissue.     

Feline calicivirus VP2 is essential for the production of infectious virions

The third open reading frame (ORF3) located at the 3′ end of the genomic RNA of feline calicivirus (FCV) encodes a small (12.2-kDa) minor structural protein of 106 amino acids designated VP2. Point mutations and deletions were introduced into an infectious FCV cDNA clone in order to evaluate the functional importance of ORF3 and its encoded protein, VP2. Deletion of the entire ORF3 sequence was lethal for the virus, and evidence was found for strong selective pressure to produce the VP2 protein. Extended deletions in the 5′ end and small deletions in the 3′ end of ORF3, as well as the introduction of stop codons into the ORF3 sequence, were tolerated by the viral replication machinery, but infectious virus could not be recovered. Infectious virus particles could be rescued from a full-length FCV cDNA clone encoding a nonfunctional VP2 when VP2 was provided in trans from a eukaryotic expression plasmid. Our data indicate that VP2, a protein apparently unique to the caliciviruses, is essential for productive replication that results in the synthesis and maturation of infectious virions and that the ORF3 nucleotide sequence itself overlaps a cis-acting RNA signal at the genomic 3′ end.