Potato tuber type H phosphorylase isozyme. Molecular cloning, nucleotide sequence, and expression of a full-length cDNA in Escherichia coli

Higher plant tissues contain two alpha-glucan phosphorylase isozymes (EC 2.4.1.1), types L and H, localized in the plastid and the cytoplasm, respectively. We already isolated and sequenced a cDNA clone encoding the type L isozyme. Presently, a cDNA clone encoding the type H counterpart was isolated from a cDNA library of immature potato tuber by plaque hybridization, using two oligonucleotide probes synthesized based on the partial amino acid sequences of the type H isozyme. The message encodes a polypeptide of 838 amino acid residues. Sequence comparison of the two potato tuber phosphorylase isozymes revealed two major distinctions; the type L isozyme contains a 78-residue insertion in the middle of the polypeptide chain as well as a 50-residue amino-terminal extension. Except for these extra portions, the two isozyme sequences show an identity of 63%. The entire structural gene for the type H isozyme was inserted 3'-downstream of the strong T7 RNA polymerase promoter in the expression plasmid pET-3b. Escherichia coli BL21 (DE3) cells carrying this plasmid produced active phosphorylase upon induction with isopropyl-beta-D-thiogalactoside at 22 degrees C. The expression is entirely dependent on the temperature; the bacteria did not produce a detectable amount of the active enzyme at 37 degrees C. Addition of pyridoxine to the culture medium was effective for the enzyme production.     

Multiple cDNA sequences and the evolution of bovine stomach lysozyme

To investigate the origin of stomach expression of lysozyme in ruminants; we surveyed clones from a cow stomach cDNA library with a lysozyme cDNA probe. Ten percent of the clones in this library were lysozyme-specific. Thirty of the lysozyme clones were sequenced, and seven types of lysozyme mRNA sequence were found. They encode the three previously identified stomach isozymes of lysozyme. The seven sequences are closely related to one another and represent the products of a minimum of 4 of the approximately 10 cow lysozyme genes detected by genomic blotting. The most abundant form of stomach lysozyme (form 2) is encoded by at least two genes, whereas forms 1 and 3 are possibly each encoded by only one gene. The number of genes encoding each isozyme appears to contribute the largest factor in the relative abundance of each isozyme. The multiple lysozyme genes expressed in the cow stomach are the result of gene duplications that occurred during ruminant evolution. The recruitment of lysozyme as a major enzyme in the stomach may thus have involved an early regulatory event and a later 4-7-fold increase in expression allowed by gene amplification. During this period, the amino acid sequences of these lysozymes have been evolving more slowly than those of nonruminant lysozymes.     

The Molecular Evolution of Actin

We have investigated the molecular evolution of plant and nonplant actin genes comparing nucleotide and amino acid sequences of 20 actin genes. Nucleotide changes resulting in amino acid substitutions (replacement substitutions) ranged from 3-7% for all pairwise comparisons of animal actin genes with the following exceptions. Comparisons between higher animal muscle actin gene sequences and comparisons between higher animal cytoplasmic actin gene sequences indicated less than 3% divergence. Comparisons between plant and nonplant actin genes revealed, with two exceptions, 11-15% replacement substitution. In the analysis of plant actins, replacement substitution between soybean actin genes SAc1, SAc3, SAc4 and maize actin gene MAc1 ranged from 8-10%, whereas these members within the soybean actin gene family ranged from 6-9% replacement substitution. The rate of sequence divergence of plant actin sequences appears to be similar to that observed for animal actins. Furthermore, these and other data suggest that the plant actin gene family is ancient and that the families of soybean and maize actin genes have diverged from a single common ancestral plant actin gene that originated long before the divergence of monocots and dicots. The soybean actin multigene family encodes at least three classes of actin. These classes each contain a pair of actin genes that have been designated kappa (SAc1, SAc6), lambda (SAc2, SAc4) and mu (SAc3, SAc7). The three classes of soybean actin are more divergent in nucleotide sequence from one another than higher animal cytoplasmic actin is divergent from muscle actin. The location and distribution of amino acid changes were compared between actin proteins from all sources. A comparison of the hydropathy of all actin sequences, except from Oxytricha, indicated a strong similarity in hydropathic character between all plant and nonplant actins despite the greater number of replacement substitutions in plant actins. These protein sequence comparisons are discussed with respect to the demonstrated and implicated roles of actin in plants and animals, as well as the tissue-specific expression of actin.     

Cytochrome c: gene structure, homology and ancestral relationships.

In this paper, the author notes the recommended definition of the word "homology" (i.e., indicating an ancestral relationship) and the recommended stipulation that "evidence for homology should be explicitly laid out". The postulated homology for somatic and testes-specific isozymes of cytochrome c is then examined, using recent data obtained from the study of cytochrome c genes. Consideration is also given to some newer findings of molecular biology and possibilities are considered for various types of change in the genome of an organism. Possible roles of introns, pseudogenes and multigene families are considered. The relationship of testes-specific cytochrome c to somatic cytochrome c is carefully considered from data obtained in experimental studies of genes of these two isozymes. If one assumes that these isozymes arose as a consequence of a gene duplication, data from rat and mouse genes indicate that the testes-specific isozyme has incorporated more amino acid changes than the somatic isozyme since the time of their divergence. However, when the 15 amino acid differences (testes-specific vs. somatic isozyme) are considered, there is virtually no similarity in these 15 positions of the testes-specific isozyme with any of the hypothetical ancestral sequences of the somatic isozyme. Nucleotide differences in cytochrome c genes have been evaluated by comparing genes for the two rodent cytochrome c isozymes to cytochrome c genes of fruit flies, chickens and humans. Comparisons of nucleotide substitution rates in genes for the two cytochrome c isozymes in rodents confirm the conclusions from amino acid sequence comparisons; namely, that more rapid nucleotide changes have occurred in the testes-specific cytochrome c gene, than in the somatic cytochrome c gene. Possible explanations for these findings are considered.     

Evolution of C4 phosphoenolpyruvate carboxylase in Flaveria, a conserved serine residue in the carboxyl-terminal part of the enzyme is a major determinant for C4-specific characteristics

C4 phosphoenolpyruvate carboxylases have evolved from ancestral C3 isoforms during the evolution of angiosperms and gained distinct kinetic and regulatory properties compared with the C3 isozymes. To identify amino acid residues and/or domains responsible for these C4-specific properties the C4 phosphoenolpyruvate carboxylase of Flaveria trinervia (C4) was compared with its orthologue in the closely related C3 plant Flaveria pringlei. Reciprocal enzyme chimera were constructed and the kinetic constants, K(0.5) and k(cat), as well as the Hill coefficient, h, were determined for the substrate phosphoenolpyruvate both in the presence and absence of the activator glucose 6-phosphate. By this approach two regions were identified which determined most of the kinetic differences of the C4 and C3 ppcA phosphoenolpyruvate carboxylases with respect to the substrate PEP. In addition, the experiments suggest that the two regions do not act additively but interact with each other. The region between amino acids 296 and 437 is essential for activation by glucose 6-phosphate. The carboxyl-terminal segment between amino acids 645 and 966 contains a C4 conserved serine or a C3 invariant alanine at position 774 in the respective enzyme isoform. Site-directed mutagenesis shows that this position is a key determinant for the kinetic properties of the two isozymes.     

Immunological analysis of the phosphorylation state of maize C4-form phosphoenolpyruvate carboxylase with specific antibodies raised against a synthetic phosphorylated peptide

The phosphoenolpyruvate carboxylase (PEPC) isozyme involved in C4 photosynthesis is known to undergo reversible regulatory phosphorylation under illuminated conditions, thereby decreasing the enzyme's sensitivity to its feedback inhibitor, L-malate. For the direct assay of this phosphorylation in intact maize leaves, phosphorylation state-specific antibodies to the C4-form PEPC were prepared. The antibodies were raised in rabbits against a synthetic phosphorylated 15-mer peptide with a sequence corresponding to that flanking the specific site of regulatory phosphorylation (Ser15) and subsequently purified by affinity-chromatography. Specificity of the resulting antibodies to the C4-form PEPC phosphorylated at Ser15 was established on the basis of several criteria. The antibodies did not react with the recombinant root-form of maize PEPC phosphorylated in vitro. By the use of these antibodies, the changes in PEPC phosphorylation state were semi-quantitatively monitored under several physiological conditions. When the changes in PEPC phosphorylation were monitored during the entire day with mature (13-week-old) maize plants grown in the field, phosphorylation started before dawn, reached a maximum by mid-morning, and then decreased before sunset. At midnight dephosphorylation was almost complete. The results suggest that the regulatory phosphorylation of C4-form PEPC in mature maize plants is controlled not only by a light signal but also by some other metabolic signal(s). Under nitrogen-limited conditions the phosphorylation was enhanced even though the level of PEPC protein was decreased. Thus there seems to be some compensatory regulatory mechanism for the phosphorylation.     

Nucleotide sequence of cDNA encoding the small subunit of ribulose-1,5-bisphosphate carboxylase from maize

We have cloned a full length cDNA for the small subunit of ribulose-1,5-bisphosphate carboxylase from C4 monocot maize, determined the complete nucleotide sequence of this cDNA and deduced its amino acid sequence. The cDNA insert included 513 bp of the coding region, and 65 and 252 nucleotides of the 5' and 3' untranslated regions, respectively. The transit and mature peptides have, respectively, 47 and 123 amino acids. Comparison with the small subunit genes from other plants revealed that the maize small subunit is similar to the wheat one, there being 73% homology between the transit peptides and 64% between the mature proteins. This indicates that there is no noteworthy difference between the C3 and C4 small subunit structures. Extreme codon bias was observed for this gene, and similar codon preferences are observed for other proteins highly expressed in maize leaf, light harvesting chlorophyll binding protein and phosphoenolpyruvate carboxylase. The results indicate that preferential codon usage for highly expressed genes occurs in maize leaf.     

Lactate dehydrogenase-B cDNA from the teleost Fundulus heteroclitus: evolutionary implications

A cDNA that encodes the heart-type lactate dehydrogenase (LDH-B) from the teleost fish Fundulus heteroclitus was cloned and sequenced. The protein encoded by the cDNA was analyzed in relation to 13 LDH proteins from a variety of taxa. One of the deductions from this analysis is that LDH-B proteins have residues in the active site that are unique and that may be important in determining the biochemistry of the heart-type isozyme. Phylogenetic analysis of the LDH sequences indicates that the branch lengths are greater in lower vertebrates, suggesting that the amino acid replacement rates vary depending on the evolutionary constraints within each taxon. Furthermore, the analysis suggests that LDH-C arose prior to the divergence of the LDH-A and LDH-B isozymes and thus that it is probably ancestral to these isozymes.     

Genetic diversity at class II DRB loci of the primate MHC

The evolution of polymorphism at loci encoding the beta-chains of the MHC class II DR Ag was studied in primates by DNA amplification (polymerase chain reaction). Phylogenetic analysis of 63 DRB sequences from the polymorphic second exon (first domain) of nonhuman primates and 53 human sequences indicates the presence of five DRB loci in primates, derived from a DRB1-like ancestral locus over 20 million yr ago. Many of the allelic types at the DRB1 locus predate the divergence of hominoids (5 million yr ago) and some (DR4, DR3, 5, 6) predate the divergence of Old world monkeys and hominoids (20 million yr ago). The DRB3 locus appears to have arisen before the divergence of hominoids on an ancestral DRB1 lineage. The DRB2 and DRB5 loci were generated more than 20 million yr ago and the DRB4 locus more than 5 million yr ago. The DRB2 locus, a pseudogene in humans, is polymorphic in the nonhuman primates.     

Characterization of a single soybean cDNA encoding cytosolic and glyoxysomal isozymes of aspartate aminotransferase

A soybean cDNA clone, pSAT1, which encodes both the cytosolic and glyoxysomal isozymes of aspartate aminotransferase (AAT; EC 2.6.1.1) was isolated. Genomic Southern blots and analysis of genomic clones indicated pSAT1 was encoded by a single copy gene. pSAT1 contained an open reading frame with ca. 90% amino acid identity to alfalfa and lupin cytosolic AAT and two in-frame start codons, designated ATG1 and ATG2. Alignment of this protein with other plant cytosolic AAT isozymes revealed a 37 amino acid N-terminal extension with characteristics of a peroxisomal targeting signal, designated PTS2, including the modified consensus sequence RL-X₅-HF. The second start codon ATG2 aligned with previously reported start codons for plant cytosolic AAT cDNAs. Plasmids constructed to express the open reading frame initiated by each of the putative start codons produced proteins with AAT activity in Escherichia coli. Immune serum raised against the pSAT1-encoded protein reacted with three soybean AAT isozymes, AAT1 (glyoxysomal), AAT2 (cytosolic), and AAT3 (subcellular location unknown). We propose the glyoxysomal isozyme AAT1 is produced by translational initiation from ATG1 and the cytosolic isozyme AAT2 is produced by translational initiation from ATG2. N-terminal sequencing of purified AAT1 revealed complete identity with the pSAT1-encoded protein and was consistent with the processing of the PTS2. Analysis of cytosolic AAT genomic sequences from several other plant species revealed conservation of the two in-frame start codons and the PTS2 sequence, suggesting that these other species may utilize a single gene to generate both cytosolic and glyoxysomal or peroxisomal forms of AAT.     

Cloning and sequence analysis of cDNA encoding aspartate aminotransferase isozymes from Panicum miliaceum L., a C4 plant.

The cytosolic and mitochondrial isozymes of aspartate aminotransferase (AspAT) function in the C4 dicarboxylate cycle of photosynthesis. We constructed a cDNA library from leaf tissues of Panicum miliaceum, an NAD-malic-enzyme-type C4 plant and screened the library for AspAT isozymes. A full-length cDNA clone for cytosolic AspAT was isolated. This clone contains an open reading frame that encodes 409 amino acids. We also isolated two cDNA clones for different precursors of mitochondrial AspAT. Comparing these two sequences in the coding regions, we found 12 amino acid substitutions out of 28 base substitutions. The encoded amino acid sequences predict that mitochondrial AspAT are synthesized as precursor proteins of 428 amino acid residues, which each consist of a mature enzyme of 400 amino acid residues and a 28-amino-acid presequence. This prediction coincides with the observation that the in vitro translation product of the mRNA for mitochondrial AspAT was substantially larger than the mature form. A comparison of the amino acid sequences of the AspAT isozymes from P. miliaceum with the published sequences for the enzymes from various animals and microorganisms reveals that functionally and/or structurally important residues are almost entirely conserved in all AspAT species.     

Maize C4 photosynthesis involves differential regulation of phosphoenolpyruvate carboxylase genes

Maize as a C4 plant partitions CO2 fixation in two consecutive, spatially separated steps, thus eliminating photorespiration. The crucial enzyme for primary CO2 fixation is a C4-specific phosphoenolpyruvate carboxylase (PEPC). The differential expression of the unique C4-specific gene pepcZm1 and two non-C4-specific genes, pepcZm2A and pepcZm3B, in leaf, root, and stem is reported here. It is shown, in a transient homologous system, that this tissue-specific regulation is mainly controlled by their distinct promoters. The light induction of the C4-specific pepcZm1 in illuminated etiolated (greening) leaves probably relies on light-dependent developmental changes instead of an immediate responsiveness found for other maize genes. Analyses of deleted, mutated, and hybrid promoters revealed the redundant nature of a 14mer which is repeated four times and a decisive function of the TATA box-like motif, TATTT, and the sequences directly preceding it. No consensus sequences to other photosynthetic gene promoters were uncovered. Although light induces the expression of C4 PEPC and other photosynthetic genes in maize, this co-ordination is apparently mediated through different signal transduction pathways and distinct regulatory elements. This study indicates that the acquisition of a new promoter is at least partially responsible for the C4-specific expression of pepcZm1 essential for C4 photosynthesis.     

The complete amino acid sequence of the human aldolase C isozyme derived from genomic clones

The complete protein sequence of the human aldolase C isozyme has been determined from recombinant genomic clones. A genomic fragment of 6673 base pairs was isolated and the DNA sequence determined. Aldolase protein sequences, being highly conserved, allowed the derivation of the sequence of this isozyme by comparison of open reading frames in the genomic DNA to the protein sequence of other human aldolase enzymes. The protein sequence of the third aldolase isozyme found in vertebrates, aldolase C, completes the primary structural determination for this family of isozymes. Overall, the aldolase C isozyme shared 81% amino acid homology with aldolase A and 70% homology with aldolase B. The comparisons with other aldolase isozymes revealed several aldolase C-specific residues which could be involved in its function in the brain. The data indicated that the gene structure of aldolase C is the same as other aldolase genes in birds and mammals, having nine exons separated by eight introns, all in precisely the same positions, only the intron sizes being different. Eight of these exons contain the protein coding region comprised of 363 amino acids. The entire gene is approximately 4 kilobases.     

Four cytosolic-type CuZn-superoxide dismutases in germinating seeds of Pinus sylvestris

A differential analysis of CuZn-superoxide dismutase (SOD. EC 1.15.1.1) isozymes after native-polyacry lamide gel elecrrophoresis (PAGE) and isoelectric focusing (IEF) indicated that germinating seeds of Scots pine (Pinus sylvestris L.) 3 days after the start of imbibition (3 DAI) contain five CuZn-SOD isozymes. Two isozymes co-migrated on native–PAGE but were separated after IEF. CuZn-SODs of Scots pine were purified from germinating seeds (3 DAI) by anion-exchange chromatography, hydrophobic interaction chromatography and chromatofocusing. The final separation of CuZn-SOD isozymes was accomplished by native-PAGE. CuZn-SOD isozymes were electroblotted and their NH₂-terminal amino acid sequence was determined. Comparisons of the amino acid sequences with sequences of CuZn-SOD isozymes from other plant sources indicated that one CuZn-SOD isozyme was of the chloroplastic type whereas the other four isozymes belonged to the cytosolic-type CuZn-SODs, The NH₂-terminal amino acid sequence of the chloroplastic CuZn-SOD and of one cytosolic-type CuZn-SOD were identical to those of two previously isolated, sequenced and localized CuZn-SOD isozymes from Scots pine needles. Two cytosolic-type CuZn-SOD isozymes showed a homology at 20 out of 21 NH₂-terminal amino acids. Mitochondria and glyoxysomes were isolated by differential and Percoll density-gradient centrifugation from germinating seeds (3 DAI). The cell fractionation experiments did not suggest that a major part of the CuZn-SOD activity in germinating seeds was derived from glyoxysomes or mitochondria.     

Molecular cloning of a cDNA for rat hepatic glutaminase. Sequence similarity to kidney-type glutaminase

Mammalian liver possesses a unique isozyme of phosphate-activated glutaminase which plays an important role in the regulation of glutamine catabolism. Antibodies to hepatic glutaminase were used to screen a lambda gt11 rat liver cDNA library. One cDNA to hepatic glutaminase was identified. Changes in the relative abundance of hepatic glutaminase mRNA were determined by hybridization to this cDNA. The mRNA is found only in liver; it is not present prior to birth but its abundance increases dramatically at birth. The abundance of the mRNA is increased approximately 4-fold in diabetes. The sequence of the cDNA was compared to that recently published for kidney (brain)-type glutaminase (Banner, C., Hwang, J.-J., Shapiro, R.A., Wenthold, R.J., Nakatani, Y., Lampel, K.A., Thomas, J.W., Huie, D., and Curthoys, N.P. (1988) Mol. Brain Res. 3, 247-254). When the predicted amino acid sequences were compared a region of 123 amino acids with greater than 80% identity was found. The presence of scattered amino acid substitutions within stretches of identical amino acids suggests that the glutaminase isozymes are encoded by separate genes. This is the first demonstration of any similarity between the two glutaminases at the molecular level.     

Maize C4-form phosphoenolpyruvate carboxylase engineered to be functional in C3 plants: mutations for diminished sensitivity to feedback inhibitors and for increased substrate affinity

Introducing a C(4)-like pathway into C(3) plants is one of the proposed strategies for the enhancement of photosynthetic productivity. For this purpose it is necessary to provide each component enzyme that exerts strong activity in the targeted C(3) plants. Here, a maize C(4)-form phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) was engineered for its regulatory and catalytic properties so as to be functional in the cells of C(3) plants. Firstly, amino acid residues Lys-835 and Arg-894 of maize PEPC, which correspond to Lys-773 and Arg-832 of Escherichia coli PEPC, respectively, were replaced by Gly, since they had been shown to be involved in the binding of allosteric inhibitors, malate or aspartate, by our X-ray crystallographic analysis of E. coli PEPC. The resulting mutant enzymes were active but their sensitivities to the inhibitors were greatly diminished. Secondly, a Ser residue (S780) characteristically conserved in all C(4)-form PEPC was replaced by Ala conserved in C(3)- and root-form PEPCs to decrease the half-maximal concentration (S(0.5)) of PEP. The double mutant enzyme (S780A/K835G) showed diminished sensitivity to malate and decreased S(0.5)(PEP) with equal maximal catalytic activity (V(m)) to the wild-type PEPC, which will be quite useful as a component of the C(4)-like pathway to be introduced into C(3) plants.