The primary structure of human H-protein of the glycine cleavage system deduced by cDNA cloning

A full-length cDNA encoding the human H-protein of the glycine cleavage system has been isolated from a lambda gt11 human fetal liver cDNA library. The cDNA insert was 1091 base pairs with an open reading frame of 519 base pairs which encoded a 125-amino acid mature human H-protein with a 48-amino acid presequence. Human H-protein is 97%, 86%, and 46% identical to the bovine, chicken, and pea H-protein, respectively.     

Molecular cloning of cDNAs for rat proteasomes: deduced primary structures of four other subunits.

Proteasomes (multi-protease complexes) are composed of approximately 15 non-identical subunits of similar sizes (molecular weight = 21-32 kDa), but different charges (isoelectric point = 4-9). Previously, we deduced the primary structures of 6 subunits of rat proteasomes by recombinant DNA techniques. In this paper we report the nucleotide sequences of 4 other subunits, rIOTA, rZETA, rDELTA, and rRING12, determined from cDNA clones isolated by screening a rat H4TG hepatoma cell cDNA library with the cDNAs of their human counterparts as probes. The polypeptides deduced from their nucleotide sequences consisted of 246, 241, 202, and 219 amino acid residues with calculated molecular weights of 27,399, 26,391, 21,649, and 23,324, and calculated isoelectric points of 6.37, 4.65, 4.84, and 4.70, respectively. These results and previous findings indicate that the primary structures of the subunits of rat proteasomes show considerably high inter-subunit homology, but can be classified into apparently distinct sub-groups, suggesting that rat proteasome genes form a multi-gene family with the same evolutionary origin, but have diverged during evolution to acquire possibly subunit-specific functions.     

Molecular cloning of cDNAs for human fibroblast nucleotide pyrophosphatase.

Nucleotide pyrophosphatase (NPPase) activity is markedly elevated in cultured skin fibroblasts from patients of Lowe's syndrome. cDNA clones encoding the NPPase were isolated using synthetic oligonucleotide probes designed on the basis of partial amino acid sequence of the enzyme purified from human placenta. The complete sequences of these clones yielded a merged sequence of 3508 bases. The polypeptide chain of the enzyme was deduced to comprise 873 amino acids with a calculated molecular weight of 99,703 and had the characteristics of a class II transmembrane protein. Ten potential N-glycosylation sites were detected in the protein. RNA blot analysis showed that human fibroblasts contain two minor mRNAs of 7.0 and 8.2 kb, respectively, in addition to a major 3.6-kb species that coincides with the merged cDNA in size. A computer search of a nucleotide sequence data-base revealed that plasma cell membrane glycoprotein PC-1, whose function was unknown at the time, is identical with the NPPase. Comparison of the amino acid sequence of the NPPase with the active site sequence of bovine 5'-nucleotide phosphodiesterase allowed the assignment of a putative active site domain to the central region of the COOH-terminal extracellular domain of the NPPase. The gene for human NPPase was localized to chromosome 6 at q22-q23 by in situ hybridization with a fragment of the NPPase cDNA.     

Molecular cloning and sequence analysis of a chicken ornithine decarboxylase cDNA

A chicken embryo cDNA library was screened with a mouse probe for ornithine decarboxylase (ODC) and 14 positively hybridizing clones isolated. The longest of these (1.7 kb) was sub-cloned and sequenced. It is estimated that the clone comprises approximately 98% of the coding region for chicken ODC. The DNA sequence shows 78% identity with the human ODC cDNA sequence and the deduced amino acid sequence is almost 90% homologous to mouse and human. Both the peptide and cDNA sequences show interesting potential regulatory features which are discussed here.     

Molecular cloning of a cDNA encoding chicken T-protein of the glycine cleavage system and expression of the functional protein in Escherichia coli. Effect of mRNA secondary structure in the translational initiation region on expression.

DNA clones encoding chicken T-protein of the glycine cleavage system were isolated from chicken liver lambda gt10 cDNA libraries. Three overlapping clones provided an open reading frame of 1176 nucleotides that predicts a polypeptide of 392 amino acids (M(r) 42,056) comprised of a 16-residue mitochondrial targeting sequence and a 376-residue mature protein (M(r) 40,292). The amino acid sequence predicted for the mature protein showed 67% identity with that of bovine T-protein. A cDNA encoding mature T-protein was constructed, and the nucleotide sequence just downstream of the initiation codon was modified without amino acid substitution to reduce the free energy of formation for the folded mRNA. Expression plasmids containing these cDNA variants produced large amounts of T-protein in Escherichia coli, while very low expression was observed with a plasmid containing wild type cDNA. Enzymatically active T-protein was obtained when the expression was conducted at 30 degrees C with 25 microM isopropyl-1-thio-beta-D-galactopyranoside. Under the full inducing condition (at 37 degrees C and 1 mM inducer), the expressed T-protein was recovered as insoluble and inactive protein. The recombinant T-protein was purified to near homogeneity with a yield of about 30%. Apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is approximately 40,000, similar to the size of T-protein purified from chicken liver. NH2-terminal amino acid sequence analysis (9 residues) revealed 100% identity with chicken T-protein determined chemically. The kinetic properties of the recombinant T-protein resembled those of the native chicken T-protein.     

Molecular phylogeny of Flaveria as deduced from the analysis of nucleotide sequences encoding the H-protein of the glycine cleavage system

Sequence comparisons have shown that nucleotide sequences of the H-protein, a component of the glycine cleavage system, are only moderately conserved and can be used as molecular markers for intrageneric phylogenetic studies. We have analysed the respective cDNA sequences from 12 species of Flaveria, and a more limited set of gdcsH upstream regions. These data are discussed with respect to a phylogenetic reconstruction of Flaveria, a small genus which includes species of different photo-synthetic types, namely C₃, C₃-C₄, C₄-like and C₄. Our analysis essentially supports an earlier hypothesis, based on morphological and eco-geographical data, of the evolution of Flaveria (Powell 1978). This close agreement shows the usefulness of H-protein nucleotide sequences at a low taxonomic level. Our analysis independently confirms that C₄ photosynthesis has evolved two times in different lineages of Flaveria. Most remarkably, the C₄ taxa of Flaveria appear as derived relative to the C₃-C₄ intermediate taxa, i.e. they probably have common direct predecessors. This is the first direct evidence for a phylo-genetically intermediate position of C₃-C₄ intermediate photosynthesis. Our data also confirm the antiquity of C₃ photosynthesis in Flaveria but suggest that the collection of F.pringlei used in our experiments, although clearly of C₃ photosynthetic metabolism, possibly originated from hybridization with a more advanced taxon.     

Stoichiometry of two plant glycine decarboxylase complexes and comparison with a cyanobacterial glycine cleavage system

The multienzyme glycine cleavage system (GCS) converts glycine and tetrahydrofolate to the one‐carbon compound 5,10‐methylenetetrahydrofolate, which is of vital importance for most if not all organisms. Photorespiring plant mitochondria contain very high levels of GCS proteins organised as a fragile glycine decarboxylase complex (GDC). The aim of this study is to provide mass spectrometry‐based stoichiometric data for the plant leaf GDC and examine whether complex formation could be a general property of the GCS in photosynthesizing organisms. The molar ratios of the leaf GDC component proteins are 1L₂‐4P₂‐8T‐26H and 1L₂‐4P₂‐8T‐20H for pea and Arabidopsis, respectively, as determined by mass spectrometry. The minimum mass of the plant leaf GDC ranges from 1550 to 1650 kDa, which is larger than previously assumed. The Arabidopsis GDC contains four times more of the isoforms GCS‐P1 and GCS‐L1 in comparison with GCS‐P2 and GCS‐L2, respectively, whereas the H‐isoproteins GCS‐H1 and GCS‐H3 are fully redundant as indicated by their about equal amounts. Isoform GCS‐H2 is not present in leaf mitochondria. In the cyanobacterium Synechocystis sp. PCC 6803, GCS proteins concentrations are low but above the complex formation threshold reported for pea leaf GDC. Indeed, formation of a cyanobacterial GDC from the individual recombinant GCS proteins in vitro could be demonstrated. Presence and metabolic significance of a Synechocystis GDC in vivo remain to be examined but could involve multimers of the GCS H‐protein that dynamically crosslink the three GCS enzyme proteins, facilitating glycine metabolism by the formation of multienzyme metabolic complexes. Data are available via ProteomeXchange with identifier PXD018211.     

Molecular cloning of a novel ras-like protein from chicken.

We have isolated a cDNA clone from a chicken DNA expression library which codes for a ras-like polypeptide of 216 amino acid residues. This polypeptide is closely related to the human protein TC4 and to the yeast protein Spil, two novel proteins that may be involved in the coordination of the cell cycle. In the amino-terminal region, the three polypeptides possess a P-loop motif characteristic of GTP-binding proteins. At the carboxy-terminal end, however, they lack the typical CAAX-box which is usually responsible for membrane anchorage of ras-like proteins. It is therefore likely that the three polypeptides define a new subclass of GTP-binding proteins within the ras-like superfamily.     

Molecular cloning of the chicken avidin cDNA.

A cDNA for chicken avidin was identified in a chicken oviduct cDNA library by screening with antibodies and synthetic oligodeoxyribonucleotides. Four recombinant clones were characterized and each contained the sequence of the oligonucleotide probes used in screening. They were capable also of expressing an antigen recognizable by a polyclonal or a mixture of monoclonal antibodies raised against avidin. The longest clone, lambda cAV4, contained the entire coding sequence of avidin along with a signal peptide of 24 amino acids. An avidin mRNA, approximately 700 nucleotides in length, was induced by a single injection of progesterone over a period of twenty four hours. The avidin mRNA was distributed in a tissue-specific manner, since detectable concentration of the mRNA appeared only in the oviduct after stimulation with progesterone alone or with a combination of progesterone and estrogen. No avidin mRNA was detected in the liver or kidney under these conditions. Preliminary results on the genomic complexity of avidin suggest a single copy gene. Isolation of the natural gene for avidin and studies on its regulation now can be initiated using the cDNA probe.     

Molecular cloning of human intestinal mucin cDNAs. Sequence analysis and evidence for genetic polymorphism

A human small intestine lambda gt11 cDNA library was screened using antisera prepared against the deglycosylated protein backbone of human colon cancer xenograft mucin. Three cDNAs were isolated from this screening, designated SMUC 40-42. These cDNAs were all found to contain tandem repeats of 69 nucleotides which encoded a threonine- and proline-rich protein consensus sequence of PTTTPITTTTTVTPTPTPTGTQT. RNA blots probed with one of these cDNAs, SMUC 41, exhibited large, polydisperse hybridization bands at approximately 7,600 bases. Band intensities were strongest when human small intestine, colon, and colon cancer poly(A)+ RNA was used. In vitro translation of poly(A)+ RNA from human small intestine, colon, and colon cancer cells produced a 162,000-dalton peptide that was immunoprecipitated with antibodies to deglycosylated mucin. SMUC 41 was also used to probe DNA blots, which indicated the presence of restriction fragment length polymorphisms in the intestinal mucin gene. These findings may be important in assessing the abnormal mucins found associated with several human diseases.     

Molecular cloning and sequencing of a novel cDNA encoding for a protein involved in human sperm function.

A cDNA encoding for an antigen, designated as NZ-3, was cloned and sequenced from human testis. The 1481-bp NZ-3 cDNA yielded an open reading frame (ORF) of 231 amino acids (aa) with the first ATG, Met start codon at nucleotide (nt) 104 and the stop codon TGA at nt 797. Extensive computer search indicated it to be a novel cDNA/protein. The ORF of NZ-3 cDNA was subcloned into pGEX-1lambdaT vector and expressed in glutathione S-transferase gene fusion system. The expressed recombinant protein had a molecular size of approximately 25 kDa, and the rabbit antibodies (Ab) against the recombinant antigen recognized a specific protein band of 63 +/- 3 kDa in the human testis extract. The NZ-3 antigen was located on the acrosomal and tail regions of human sperm cell and the NZ-3 Ab significantly (P < 0.001) inhibited human sperm capacitation and/or acrosome reaction. The novel recombinant NZ-3 antigen may find applications in immunocontraception and in specific diagnosis of human infertility.     

The lpd gene product functions as the L protein in the Escherichia coli glycine cleavage enzyme system.

The lpd-encoded lipoamide dehydrogenase, common to the pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes, also functions as the lipoamide dehydrogenase (L protein) in the Escherichia coli glycine cleavage (GCV) enzyme complex. Inducible GCV enzyme activity was not detected in an lpd deletion mutant; lpd+ transductants had normal levels of inducible GCV enzyme activity. A serA lpd double mutant was unable to utilize glycine as a serine source and lacked detectable GCV enzyme activity, the phenotype of a serA gcv mutant. Transformation of the double mutant with a plasmid encoding a functional lpd gene restored the ability of the mutant to use glycine as a serine source and restored inducible GCV enzyme activity to normal levels. The presence of acetate and succinate in the growth medium of a strain wild type for lpd and gcv resulted in a 50% reduction in inducible GCV enzyme activity. Enzyme levels were restored to normal under these growth conditions when the strain was transformed with a plasmid encoding a functional lpd gene.