The nucleotide sequence of a cDNA clone containing the entire coding region for mouse X-chromosome-linked phosphoglycerate kinase

A clone containing cDNA for X chromosome-linked phosphoglycerate kinase (PGK-1) was isolated from a mouse myeloma cDNA library. The nucleotide (nt) sequence of the cDNA has been determined, and the amino acid (aa) sequence of the enzyme thereby deduced. At the nt level, the coding region of mouse PGK cDNA has 93% homology with human X-linked cDNA and 60% homology with the yeast gene. Mouse PGK-1 protein contains 416 aa and is 98%, 96% and 64% homologous with human, horse, and yeast enzyme sequences, respectively.     

Processing Pisum sativum seed storage protein precursors in vitro

The profile of polypeptides separated by SDS-PAGE from seed of major crop species such as pea(Pisum sativum) is complex,resulting from cleavage (processing) of precursors expressed from multiple copies of genes encoding vicilin and legumin,the major storage globulins.Translation in vitro of mRNAs hybridselected from mid-maturation pea seed RNAs by defined vicilin and legumin cDNA clones provided precursor molecules that were cleaved in vitro by a cell-free protease extract obtained from similar stage seed;the derived polypeptides were of comparable sizes to those observed in vivo.The feasibility of transcribing mRNA in vitro from a cDNA clone and cleavage in vitro of the derived translation products was established for a legumin clone,providing a method for determining polypeptide products of an expressed sequence.This approach will also be useful for characterising cleavage site requirements since modifications an readily be introduced at the DNA level.     

Molecular cloning of a human gastric lipase and expression of the enzyme in yeast

The molecular cloning of a cDNA coding for human gastric lipase and its expression in yeast is described. A lipase present in human gastric aspirates was purified and its N-terminal amino-acid sequence was determined. This was found to be homologous with the N-terminal sequence of rat lingual lipase. A cDNA library was constructed from mRNA isolated from human stomach tissue and probed with cloned rat lingual lipase DNA. One clone, pGL17, consisting of approximately 1450 base-pairs, contained the entire coding sequence for a human gastric lipase. The amino-acid sequence from the isolated protein and the DNA sequence obtained from the cloned gene indicated that human gastric lipase consists of a 379 amino acid polypeptide with an unglycosylated Mr of 43,162. Human gastric lipase and rat lingual lipase amino-acid sequences were closely homologous but were unrelated to porcine pancreatic lipase apart from a 6 amino-acid sequence around the essential Ser-152 of porcine pancreatic lipase. A yeast expression plasmid containing the phosphoglycerate kinase promoter and terminator sequences together with the human gastric lipase gene was constructed. Yeast transformed with this vector synthesised the lipolytically active enzyme.     

Cloning and sequencing of a cDNA encoding 2,3-bisphosphoglycerate-independent phosphoglycerate mutase from maize. Possible relationship to the alkaline phosphatase family.

The primary sequence of maize 2,3-bisphosphoglycerate-independent phosphoglycerate mutase was deduced from cDNAs isolated from maize cDNA libraries by screening with specific antibodies to the cofactor-independent enzyme and from a maize genomic clone. The genomic clone provided the 5'-nucleotide sequence encoding the N-terminal amino acids which could not be obtained from the cDNA. Confirmation that the nucleotide sequence was for the cofactor-independent phosphoglycerate mutase was obtained by sequencing the peptides generated from cyanogen bromide cleavage of the purified protein. This is the first report of the amino acid sequence of a 2,3-bisphosphoglycerate cofactor-independent phosphoglycerate mutase, which consists of 559 amino acids and is twice the molecular size of the mammalian cofactor-dependent enzyme subunit. Analysis of the cofactor-independent phosphoglycerate mutase amino acid sequence revealed no identity with the cofactor-dependent mutase types. Northern blot analysis confirmed this difference since the maize cofactor-independent phosphoglycerate mutase cDNA did not hybridize with mRNA of the cofactor-dependent mutase. The lack of amino acid identity between cofactor-dependent and -independent enzymes is consistent with their different catalytic mechanisms and suggests that both enzymes are unrelated evolutionarily and arose from two independent ancestral genes. However, a constellation of residues which are involved in metal ion binding in various alkaline phosphatases is conserved in the maize cofactor-independent phosphoglycerate mutase, which suggests that the enzyme is a member of the alkaline phosphatase family of enzymes.     

The vicilin gene family of pea (Pisum sativum L.): a complete cDNA coding sequence for preprovicilin

A cDNA plasmid bank has been constructed using mRNA from developing pea seeds and three cDNAs coding for vicilin polypeptides have been selected. These cDNAs have been sequenced and between them cover the whole of the coding sequence plus part of the 5' and 3' untranslated regions. Comparison with amino acid sequence data from the protein indicates that vicilin is synthesised as preprovicilin with subsequent removal of a signal peptide and a C-terminal peptide as well as post translational endo-proteolytic cleavage. The cDNAs represent two different classes of vicilin genes whilst amino acid data show that there are at least three major classes of vicilin polypeptide. The vicilin sequences show extensive homology with conglycinin and phaseolin except in the regions of the internal proteolytic cleavages. The evolutionary significance of this relationship is discussed.     

Nucleoside diphosphate kinase from pea chloroplasts: purification,cDNA cloning and import into chloroplasts

Nucleoside diphosphate kinase (NDPK; EC 2.7.4.6) was enriched 1900-fold from purified pea (Pisum sativum L. cv. Golf.) chloroplasts. The active enzyme preparation contained two polypeptides of apparent molecular weight 18.5 kDa and 17.4kDa. Both proteins were enzymatically active and were recognized by an antiserum raised against NDPK from spinach chloroplasts, suggesting the existence of two isoforms in pea chloroplasts. The N-terminal protein sequence data were obtained for both polypeptides and compared with the nucleotide sequence of a cDNA clone isolated from a pea cDNA library. The analysis revealed that the two NDPK forms are encoded for by one mRNA, indicating that the lower-molecular-weight form could represent a proteolytic breakdown product of the 18.5-kDa NDPK. The pea chloroplastic NDPK is made as a larger precursor protein which is imported into chloroplasts. The NDPK precursor is then processed by the stromal processing peptidase to yield the 18.5-kDa form.Abbreviations NDPK nucleoside diphosphate kinase - preNDPK precursor NDPK - ps-NDPK cDNA coding for Pisum sativum NDPK II We thank Dr. Schmidt, University Göttingen, Germany, for doing the protein sequencing. This work was supported in part by grants from the Deutsche Forschungsgemeinschaft.     

Sequence of rat skeletal muscle phosphoglycerate mutase cDNA

A cDNA clone coding rat skeletal muscle phosphoglycerate mutase was isolated from a rat muscle lambda gt10 cDNA library and its sequence was determined. The deduced protein possesses 252 amino acids and is 94% homologous with respect to human muscle phosphoglycerate mutase. No amino acids changes occur at the active site and structural predictions suggest strong conformational homologies with other enzymes of the mutase family.     

Convicilin mRNA from pea (Pisum sativum L.) has sequence homology with other legume 7S storage protein mRNA species.

Nucleotide-sequence analysis of a complementary-DNA clone for convicilin, one of the storage proteins from pea (Pisum sativum L.) seeds, shows it to be homologous with the 7S legume seed storage proteins vicilin, conglycinin and phaseolin. Convicilin is more similar to vicilin than to phaseolin or to conglycinin. Significant areas of sequence difference are discussed.     

The complete amino acid sequence of yeast phosphoglycerate kinase.

The complete amino acid sequence of yeast phosphoglycerate kinase, comprising 415 residues, was determined. The sequence of residues 1-173 was deduced mainly from nucleotide sequence analysis of a series of overlapping fragments derived from the relevant portion of a 2.95-kilobase endonuclease-HindIII-digest fragment containing the yeast phosphoglycerate kinase gene. The sequence of residues 174-415 was deduced mainly from amino acid sequence analysis of three CNBr-cleavage fragments, and from peptides derived from these fragments after digestion by a number of proteolytic enzymes. Cleavage at the two tryptophan residues with o-iodosobenzoic acid was also used to isolate fragments suitable for amino acid sequence analysis. Determination of the complete sequence now allows a detailed interpretation of the existing high-resolution X-ray-crystallographic structure. The sequence -Ile-Ile-Gly-Gly-Gly- occurs twice in distant parts of the linear sequence (residues 232-236 and 367-371). Both these regions contribute to the nucleoside phosphate-binding site. A comparison of the sequence of yeast phosphoglycerate kinase reported here with the sequences of phosphoglycerate kinase from horse muscle and human erythrocytes shows that the yeast enzyme is 64% identical with the mammalian enzymes. The yeast has strikingly fewer methionine, cysteine and tryptophan residues.     

Synthesis and secretion of wheat alpha-amylase in Saccharomyces cerevisiae

A wheat alpha-amylase cDNA clone has been fused to the phosphoglycerate kinase initiator methionine to enable synthesis in the yeast Saccharomyces cerevisiae of an alpha-amylase enzyme that is identical in size to the wild-type alpha-amylase. The alpha-amylase is synthesized with an N-terminal plant signal peptide which is recognized in the yeast host, leading to efficient processing and secretion into the medium. The secretion of alpha-amylase into the medium is quite efficient in rich medium, but barely detectable in a minimal medium.     

A pea nuclear protein that is induced by dehydration belongs to the vicilin superfamily.

The purification to homogeneity of p16, a protein with an electrophoretic mobility compatible with an apparent molecular mass of 16 kDa, from nuclei of ungerminated pea embryonic axes is described. A cDNA clone of its gene, which was designated psp54, was also isolated. The psp54 cDNA contains an open reading frame coding for a 54.4-kDa polypeptide (p54). p16 corresponds to the C-terminal third of p54, although the mechanisms by which the primary polypeptide could be processed are not yet known. The sequence of p54 is 60% identical with that of the precursor of a sucrose-binding soybean protein, and, to a lesser extent (31-34%), it shares homology with some storage proteins. p16 is also 30% homologous with Nhp2p, a yeast nuclear protein. The psp54 gene, present in a single copy in pea genome, starts being expressed during seed desiccation. Soon after rehydration in seed germination, p54 mRNA disappears and is no longer detectable in vegetative tissues, except in response to hydric stress (exposure to abscisic acid, osmolites or desiccation). p16 can be recovered from nuclei cross-linked to histone H3, when the disulfide bridges that occur in vivo are preserved. On the other hand, p16 shares some properties with dehydrins, which are thought to protect cellular structures against desiccation. We propose that the possible precursor polypeptide p54 belongs to the vicilin superfamily, members of which play a variety of roles. The function of p16 may be related to the protection of chromatin structure against desiccation during seed development.     

A higher plant mitochondrial homologue of the yeast m-AAA protease. Molecular cloning,localization, and putative function

Mitochondrial AAA metalloproteases play a fundamental role in mitochondrial biogenesis and function. They have been identified in yeast and animals but not yet in plants. This work describes the isolation and sequence analysis of the full-length cDNA from the pea (Pisum sativum) with significant homology to the yeast matrix AAA (m-AAA) protease. The product of this clone was imported into isolated pea mitochondria where it was processed to its mature form (PsFtsH). We have shown that the central region of PsFtsH containing the chaperone domain is exposed to the matrix space. Furthermore, we have demonstrated that the pea protease can complement respiration deficiency in the yta10 and/or yta12 null yeast mutants, indicating that the plant protein can compensate for the loss of at least some of the important m-AAA functions in yeast. Based on biochemical experiments using isolated pea mitochondria, we propose that PsFtsH-like m-AAA is involved in the accumulation of the subunit 9 of the ATP synthase in the mitochondrial membrane.     

A Brassica cDNA clone encoding a bifunctional hydroxymethylpyrimidine kinase/thiamin-phosphate pyrophosphorylase involved in thiamin biosynthesis

We report the characterization of a Brassica napus cDNA clone (pBTH1) encoding a protein (BTH1) with two enzymatic activities in the thiamin biosynthetic pathway, thiamin-phosphate pyrophosphorylase (TMP-PPase) and 2-methyl-4-amino-5-hydroxymethylpyrimidine-monophosphate kinase (HMP-P kinase). The cDNA clone was isolated by a novel functional complementation strategy employing an Escherichia coli mutant deficient in the TMP-PPase activity. A biochemical assay showed the clone to confer recovery of TMP-PPase activity in the E. coli mutant strain. The cDNA clone is 1746 bp long and contains an open reading frame encoding a peptide of 524 amino acids. The C-terminal part of BTH1 showed 53% and 59% sequence similarity to the N-terminal TMP-PPase region of the bifunctional yeast proteins Saccharomyces THI6 and Schizosaccharomyces pombe THI4, respectively. The N-terminal part of BTH1 showed 58% sequence similarity to HMP-P kinase of Salmonella typhimurium. The cDNA clone functionally complemented the S. typhimurium and E. coli thiD mutants deficient in the HMP-P kinase activity. These results show that the clone encodes a bifunctional protein with TMP-PPase at the C-terminus and HMP-P kinase at the N-terminus. This is in contrast to the yeast bifunctional proteins that encode TMP-PPase at the N-terminus and 4-methyl-5-(2-hydroxyethyl)thiazole kinase at the C-terminus. Expression of the BTH1 gene is negatively regulated by thiamin, as in the cases for the thiamin biosynthetic genes of microorganisms. This is the first report of a plant thiamin biosynthetic gene on which a specific biochemical activity is assigned. The Brassica BTH1 gene may correspond to the Arabidopsis TH-1 gene.     

Complementation of Saccharomyces cerevisiae auxotrophic mutants by Arabidopsis thaliana cDNAs

An Arabidopsis thaliana cDNA bank has been constituted in a Saccharomyces cerevisiae expression vector based on the phosphoglycerate kinase (PGK) promoter and terminator. This bank was used to complement eight S. cerevisiae auxotrophic markers. All of them were corrected. These results confirm the quality of the bank and the feasibility of cloning plant genes by yeast mutant complementation. The cDNA complementing the ura1 yeast mutant was sequenced, analysed and shown to encode a dihydroorotic (DHO) dehydrogenase sequence.     

Cloning and expression of a Xenopus gene that prevents mitotic catastrophe in fission yeast

In fission yeast the Weel kinase and the functionally redundant Mikl kinase provide a regulatory mechanism to ensure that mitosis is initiated only after the completion of DNA synthesis. Yeast in which both Weel and Mik1 kinases are defective exhibit a mitotic catastrophe phenotype, presumably due to premature entry into mitosis. Because of the functional conservation of cell cycle control elements, the expression of a vertebrate weel or mikl homolog would be expected to rescue such lethal mutations in yeast. A Xenopus total ovary cDNA library was constructed in a fission yeast expression vector and used to transform a yeast temperature-dependent mitotic catastrophe mutant defective in both weel and mikl. Here we report the identification of a Xenopus cDNA clone that can rescue several different yeast mitotic catastrophe mutants defective in Weel kinase function. The expression of this clone in a weel/mikl-deficient mutant causes an elongated cell phenotype under non-permissive growth conditions. The 2.0 kb cDNA clone contains an open reading frame of 1263 nucleotides, encoding a predicted 47 kDa protein. Bacterially expressed recombinant protein was used to raise a polyclonal antibody, which specifically recognizes a 47 kDa protein from Xenopus oocyte nuclei, suggesting the gene encodes a nuclear protein in Xenopus. The ability of this cDNA to complement mitotic catastrophe mutations is independent of Weel kinase activity.     

Cloning and expression of a Xenopus gene that prevents mitotic catastrophe in fission yeast

In fission yeast the Weel kinase and the functionally redundant Mikl kinase provide a regulatory mechanism to ensure that mitosis is initiated only after the completion of DNA synthesis. Yeast in which both Weel and Mik1 kinases are defective exhibit a mitotic catastrophe phenotype, presumably due to premature entry into mitosis. Because of the functional conservation of cell cycle control elements, the expression of a vertebrate weel or mikl homolog would be expected to rescue such lethal mutations in yeast. A Xenopus total ovary cDNA library was constructed in a fission yeast expression vector and used to transform a yeast temperature-dependent mitotic catastrophe mutant defective in both weel and mikl. Here we report the identification of a Xenopus cDNA clone that can rescue several different yeast mitotic catastrophe mutants defective in Weel kinase function. The expression of this clone in a weel/mikl-deficient mutant causes an elongated cell phenotype under non-permissive growth conditions. The 2.0 kb cDNA clone contains an open reading frame of 1263 nucleotides, encoding a predicted 47 kDa protein. Bacterially expressed recombinant protein was used to raise a polyclonal antibody, which specifically recognizes a 47 kDa protein from Xenopus oocyte nuclei, suggesting the gene encodes a nuclear protein in Xenopus. The ability of this cDNA to complement mitotic catastrophe mutations is independent of Weel kinase activity.     

Retion of human apolipoprotein A-II by a mutant of Saccharomyces cerevisiæ

Summary A modified cDNA of mature human apolipoprotein A-II (apoA-II) was expressed by a sterol-uptake yeast strain (erg 10) of Saccharomyces cerevisiæ. ApoA-II cDNA was fused with a modified yeast alpha factor leader peptide coding sequence under phosphoglycerate kinase promoter control in a 2 micron-based plasmid construction. The use of cholesterol allowed apoA-II secretion in the culture medium. The mono and dimeric forms of apoA-II (approximately 3 micrograms per ml) were detected.     

Use of synthetic oligodeoxyribonucleotides and primed cDNA as probes for pea (Pisum sativum L.) RNA and genomic DNA sequences

Two oligonucleotide sequences were synthesised by a solid-phase phosphotriester method. One of these sequences, A was a copy of part of a characterised cDNA clone encoding the basic subunit of legumin, a seed storage protein of Pisum sativum L. (garden pea); the other sequence B was predicted to be complementary to the 5 region of legumin mRNA on the basis of the amino acid sequence of legumin acidic subunits and most likely codon usage. Sequence A was shown to hybridise specifically to a legumin cDNA clone and to legumin mRNA. Sequence B did not hybridise specifically to legumin mRNA and was concluded not to be correctly complementary to legumin mRNA. Sequence A was used as a primer for cDNA synthesis using pea seed mRNA as a template. The cDNA so produced hybridised specifically to a legumin cDNA clone, to legumin mRNA, and to sequences encoding legumin in a restriction digest of pea genomic DNA. It is suggested that such oligonucleotide primed cDNAs may be of general value in probing eukaryotic genomic DNA.