Calmodulin isoforms in Arabidopsis encoded by multiple divergent mRNAs

Three new, unique cDNA sequences encoding isoforms of calmodulin (CaM) were isolated from an Arabidopsis cDNA library cloned in gt10. These sequences (ACaM-4, -5, and -6) represent members of the Arabidopsis CaM gene family distinct from the three DNA sequences previously reported. ACaM-4 and -6 encode full-length copies of CaM mRNAs of ca. 0.75 kb. The ACaM-5 sequence encodes a partial length copy of CaM mRNA that is lacking sequences encoding the amino-terminal 10 amino acids of mature CaM and the initiator methionine. The derived amino acid sequence of ACaM-5 is identical to the sequences encoded by two of the previously characterized ACaM cDNAs, and is identical to TCH-1 mRNA, whose accumulation was increased by touch stimulation. The polypeptides encoded by ACaM-4 and -6 differ from that encoded by ACaM-5 by six and two amino acid substititions, respectively. Most of the deduced amino acid sequence substitutions in the Arabidopsis CaM isoforms occurred in the fourth Ca²⁺-binding domain. Polymerase chain reaction amplification assays of ACaM-4, -5 and -6 mRNA sequences indicated that each accumulated in Arabidopsis leaf RNA fractions, but only ACaM-4 and -5 mRNAs were detected in silique total RNA. The six different CaM cDNA sequences each hybridize with unique Eco RI restriction fragments in genomic Southern blots of Arabidopsis DNA, indicating that these sequences were derived from distinct structural genes. Our results suggest that CaM isoforms in Arabidopsis may have evolved to optimize the interaction of this Ca²⁺-receptor protein with specific subsets of response elements.     

Cloning of cDNA Sequences Encoding the Calcium-Binding Protein, Calmodulin, from Barley (Hordeum vulgare L.)

Full- and partial-length cDNAs encoding calmodulin mRNA have been cloned and sequenced from barley (Hordeum vulgare L.). Barley leaf mRNA, size-fractionated in sucrose density gradients, was used to synthesize double-stranded cDNA. The cDNA was cloned in λgt10 and screened with a synthetic, 14-nucleotide oligonucleotide probe, which was designed using the predicted coding sequences of the carboxy termini of spinach and wheat calmodulin proteins. The primary structure of barley calmodulin, predicted from DNA sequencing experiments, consists of 148 amino acids and differs from that of wheat calmodulin in only three positions. In two of the three positions, the amino acid changes are conservative, while the third change consists of an apparent deletion/insertion. The overall nucleotide sequence similarity between the amino acid coding regions of barley and vertebrate calmodulin mRNAs is approximately 77%. However, a region encoding 11 amino acids of the second Ca²⁺-binding domain is very highly conserved at the nucleotide level compared with the rest of the coding sequences (94% sequence identity between barley and chicken calmodulin mRNAs). Genomic Southern blots reveal that barley calmodulin is encoded by a single copy gene. This gene is expressed as a single size class of mRNA in all tissues of 7-day-old barley seedlings. In addition, these analyses indicate that a barley calmodulin cDNA coding region subclone is suitable as a probe for isolating calmodulin genes from other plants.     

Isolation,sequencing and analysis of the expression of Bryonia calmodulin after mechanical perturbation

A cDNA clone (Bc329) encoding calmodulin was isolated from a Bryonia cDNA library by screening with cloned Arabidopsis calmodulin cDNA. The cDNA Bc329 was 899 bp full-length clone. The predicted amino acid sequence consists of 149 residues and reveals a high homology with other known plant calmodulins (91 to 99% identity). Genomic southern blot suggests that Bryonia calmodulin is encoded by a single-copy gene. The Bc329 clone was used as a probe to study the expression of calmodulin mRNA after a mechanical stimulus applied on young Bryonia internodes. The steady-state of calmodulin mRNA reached a maximum 30 min after the treatment before it progressively decreased. The role of calcium and calmodulin as second messengers is discussed with regard to environmental changes.     

A cDNA clone encoding Brassica calmodulin

A 834 bp cDNA encoding calmodulin (CaM) has been isolated from Brassica juncea. On Northern analysis this cDNA hybridises this cDNA to mRNAs of about 0.9 kb in leaf, silique and peduncle. Genomic Southern analysis indicates the presence of a CaM multigene family in Brassica juncea. Comparison of the predicted amino acid sequence of Brassica CaM with that of Arabidopsis CaM ACaM-2 and ACaM-3 showed 100% homology, which is not unusual, since both plants belong to the family Cruciferae. In situ hybridisation studies on Brassica seedlings using a digoxigenin-labelled RNA probe showed that high levels of CaM mRNA were detected in the leaf primordia and the shoot apical meristem, and to a lesser degree, in the zone of root elongation of the root tip. The occurrence of a higher rate of cell division and growth in these regions than its surrounding tissue may possibly be related to higher levels of CaM mRNA.     

Molecular cloning and the nucleotide sequence of cDNA for embryonic chicken pepsinogen: phylogenetic relationship with prochymosin

Embryonic chicken pepsinogen is an aspartyl proteinase that is specifically secreted during the embryonic period in the chicken proventriculus (glandular stomach). To learn the phylogeny of this pepsinogen, we isolated a cDNA clone by screening a lambda gt11 library of embryonic proventricular cDNAs with an antiserum to the embryonic chicken pepsinogen. We obtained a 200-base pair cDNA clone which encoded 18 amino acids that had high sequence homology with the carboxyl termini of other pepsinogens. Northern blot analysis revealed that this cDNA clone hybridized to a mRNA of 1,600 bases in the embryonic proventriculus but not to the mRNA in the adult proventriculus. The almost complete nucleotide sequence of embryonic chicken pepsinogen-cDNA was determined by sequencing longer cDNAs obtained by screening the same library with the 200-base pair cDNA and primer extension with a synthetic primer. The cDNA consisted of 1,281 nucleotides and encoded 383 amino acids for prepepsinogen. The predicted amino acid sequence was compared with the sequences of other aspartyl proteinases: pepsinogen A of human, monkey, pig, and chicken, progastricsin of monkey and rat, and bovine prochymosin. The phylogenetic tree constructed for them indicates the possibility that embryonic chicken pepsinogen diverged from prochymosin, after prochymosin and pepsinogen A had diverged from each other.     

Isolation of an Arabidopsis cDNA sequence encoding a 22 kDa calcium-binding protein (CaBP-22) related to calmodulin

Complementary DNA sequences were isolated from a library of cloned Arabidopsis leaf mRNA sequences in gt10 that encoded a 21.7 kDa polypeptide (CaBP-22), which shared 66% amino acid sequence identity with Arabidopsis calmodulin. The putative Ca²⁺-binding domains of CaBP-22 and calmodulin, however, were more conserved and shared 79% sequence identity. Ca²⁺ binding by CaBP-22, which was inferred from its amino acid sequence similarity with calmodulin, was demonstrated indirectly by Ca²⁺-induced mobility shifting of in vitro translated CaBP-22 during SDS-polyacrylamide gel electrophoresis. CaBP-22 is encoded by a ca. 0.9 kb mRNA that was detected by northern blotting of leaf poly(A)⁺ RNA; this mRNA was slightly larger than the 809 bp CaBP-22 cDNA insert, indicating that the deduced amino acid sequence of CaBP-22 is near full-length. CaBP-22 mRNA was detected in RNA fractions isolated from leaves of both soil-grown and hydroponically grown Arabidopsis, but below the limits of detection in RNA isolated from roots, and developing siliques. Thus, CaBP-22 represents a new member of the EF-hand family of Ca²⁺-binding proteins with no known animal homologue and may participate in transducing Ca²⁺ signals to a specific subset of response elements.     

The 5′ untranslated region of Arabidopsis thaliana calmodulin cDNA is an independent cDNA containing an open reading frame

A cDNA clone, pAUK1, with an open reading frame (ORF) coding for a hypothetical 164-amino-acid protein was isolated from an Arabidopsis thaliana (L.) Heynh cDNA library. The clone was attached, tail to tail, to the 3′ end of A. thaliana hexokinase cDNA. An almost identical sequence had been previously described as the 5′ untranslated region (5′ UTR) of A. thaliana calmodulin cDNA (ACaM-2). Sequence comparison with three additional A. thaliana truncated cDNA clones which appear in a database (GenBank) supports the conclusion that pAUKl is identical to the 5′ UTR of ACaM-2 and that the 5′ UTR of ACaM-2 is an independent cDNA artificially linked to A. thaliana calmodulin cDNA.     

A cDNA encoding 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Solanum tuberosum L

A cDNA encoding potato (Solanum tuberosum L.) 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, the first enzyme of the shikimate pathway, was cloned into phage lambda gt11. The clone represents the first cDNA for this enzyme from any eukaryotic source. The nucleotide sequence of the cDNA was determined, and its identity was confirmed through partial amino acid sequence analysis of the encoded enzyme. The cDNA contains a 1527-base pair open reading frame that encodes a polypeptide with a calculated molecular weight of 56,153. The amino terminus of the deduced polypeptide resembles a chloroplast transit sequence. Amino acid sequence identities between the mature potato enzyme and the homologous isoenzymes from Escherichia coli are only about 22%. The potato cDNA hybridized to various plant mRNAs that are all about 2 kilobases in size.     

Isolation and characterization of a cDNA clone encoding rat nucleoside diphosphate kinase

A cDNA clone for cytosolic nucleoside diphosphate (NDP) kinase was isolated from a cDNA library of rat skeletal muscle using synthetic oligonucleotides as probes. The clone constitutes a 621-base pair cDNA sequence including the 456-base pair coding region and 137-base pair 3'-untranslated one with polyadenylation site. The complete primary structure of NDP kinase was deduced from the coding sequence. An NH2-terminal amino acid sequence analysis suggested that the translated enzyme protein suffered proteolytic cleavage followed by modification at the alpha-NH2 group of the newly produced NH2-terminal amino acid residue. Taking this into account, it was tentatively concluded that the mature NDP kinase consists of 147 amino acid residues with a molecular weight of 16,724. Northern blot hybridization analysis showed that NDP kinase mRNA could be detected in total RNA fractions of brain, spleen, heart, lung, liver, kidney, testis as well as skeletal muscle, and that there was no difference in the size of mRNAs from these tissues. Tissue distribution of the mRNA nearly paralleled those of protein moiety and activity of the enzyme.     

Molecular cloning and nucleotide sequence of cDNA encoding human muscle glycogen debranching enzyme.

cDNA comprising the entire length of the human muscle glycogen debranching enzyme was cloned and its nucleotide sequence determined. The debrancher mRNA includes a 4545-base pair coding region and a 2371-base pair 3'-nontranslated region. The calculated molecular mass of the debrancher protein derived from cDNA sequence is 172,614 daltons, consistent with the estimated size of purified protein (Mr 165,000 +/- 500). A partial amino acid sequence (13 internal tryptic peptides with a total of 213 residues) determined on peptides derived from purified porcine muscle debrancher protein confirmed the identity of the cDNA clone. Comparison of the amino acid sequence predicted from the human glycogen debrancher cDNA with the partial protein sequence of the porcine debrancher revealed a high degree (88%) of interspecies sequence identity. RNA blot analysis showed that debrancher mRNA in human muscle, lymphoblastoid cells, and in porcine muscle are all similar in size (approximately 7 kilobases). Two patients with inherited debrancher deficiency had a reduced level of debrancher mRNA, whereas two other patients had no detectable abnormality in RNA blots. The isolation of the debrancher cDNA and determination of its primary structure is an important step toward defining the structure-function relationship of this multifunctional enzyme and in understanding the molecular basis of the type III glycogen storage disease.     

Separate nuclear genes encode sarcomere-specific and ubiquitous human mitochondrial creatine kinase isoenzymes

Creatine kinase (EC 2.7.3.2) isoenzymes play a central role in energy transduction. Nuclear genes encode creatine kinase subunits from muscle, brain, and mitochondria (MtCK). We have recently isolated a cDNA clone encoding MtCK from a human placental library which is expressed in many human tissues (Haas, R. C., Korenfeld, C., Zhang, Z., Perryman, B., Roman, D., and Strauss, A. W. (1989) J. Biol. Chem. 264, 2890-2897). With nontranslated and coding region probes, we demonstrated by RNA blot analysis that the MtCK mRNA in sarcomeric muscle is distinct from this placenta-derived, ubiquitous MtCK cDNA. To compare these different mRNAs, a MtCK cDNA clone was isolated from a human heart library and characterized by complete nucleotide sequence analysis. The chemically determined NH2-terminal 26 residues of purified human heart MtCK protein are identical to those predicted from this sarcomeric MtCK cDNA. The human sarcomeric and ubiquitous cDNAs share 73% nucleotide and 80% predicted amino acid sequence identities, but have less than 66% identity with the cytosolic creatine kinases. The sarcomeric MtCK cDNA encodes a 419-amino acid protein which contains a 39-residue transit peptide essential for mitochondrial import. Primer extension analysis predicts a 348-base pair 5'-nontranslated region. RNA blot analysis demonstrates that heart-derived MtCK is sarcomere-specific, but the ubiquitous MtCK mRNA is expressed in most tissues. Thus, separate nuclear genes encode two closely related, tissue-specific isoenzymes of MtCK. Our finding that multiple genes encode different mitochondrial protein isoenzymes is rare.     

The analysis of a chicken myosin heavy chain cDNA clone

A cDNA library has been constructed in the plasmid pBR322 using a large size class of RNA derived from chicken embryonic leg muscle as the template material. A clone containing a 2350-base pair insert was selected and identified as coding for the myosin heavy chain sequence, based upon its ability to hybridize to genomic myosin heavy chain clones, and by direct nucleotide sequencing. Cross-hybridization experiments with myosin heavy chain genomic clones, and mRNAs derived from different muscle types were used to explore the heterogeneity of the various myosin heavy chain isoforms at the level of the coding sequences. Although extensive sequence homology with the other isoforms was observed, a fast white isoform-specific subclone was constructed, and used to demonstrate that different genes code for the adult and embryonic fast white myosin heavy chain proteins.     

Induction of Specific mRNAs in Cultured Soybean Cells during Cytokinin or Auxin Starvation

We report the isolation of five cDNA clones whose corresponding mRNAs accumulate in cultured soybean cells (Glycine max cv Mandarin) during cytokinin or auxin starvation. The levels of three of these mRNAs decrease rapidly after addition of 5 micromolar zeatin to cytokinin-starved cells or after addition of 10 micromolar α-naphthaleneacetic acid to auxin-starved cells. These mRNAs also exhibit various patterns of accumulation in the tissues of intact soybean plants. Partial nucleotide sequence analysis demonstrates that one of the cDNAs in the collection, called SAM46, is 46% identical at the amino acid level to the iron superoxide dismutase gene of Escherichia coli. Expression of this cDNA in Escherichia coli cells results in detectable iron superoxide dismutase activity, confirming the identity of the cDNA.