Identification of Human N-Myristoylated Proteins from Human Complementary DNA Resources by Cell-Free and Cellular Metabolic Labeling Analyses

To identify physiologically important human N-myristoylated proteins, 90 cDNA clones predicted to encode human N-myristoylated proteins were selected from a human cDNA resource (4,369 Kazusa ORFeome project human cDNA clones) by two bioinformatic N-myristoylation prediction systems, NMT-The MYR Predictor and Myristoylator. After database searches to exclude known human N-myristoylated proteins, 37 cDNA clones were selected as potential human N-myristoylated proteins. The susceptibility of these cDNA clones to protein N-myristoylation was first evaluated using fusion proteins in which the N-terminal ten amino acid residues were fused to an epitope-tagged model protein. Then, protein N-myristoylation of the gene products of full-length cDNAs was evaluated by metabolic labeling experiments both in an insect cell-free protein synthesis system and in transfected human cells. As a result, the products of 13 cDNA clones (FBXL7, PPM1B, SAMM50, PLEKHN, AIFM3, C22orf42, STK32A, FAM131C, DRICH1, MCC1, HID1, P2RX5, STK32B) were found to be human N-myristoylated proteins. Analysis of the role of protein N-myristoylation on the intracellular localization of SAMM50, a mitochondrial outer membrane protein, revealed that protein N-myristoylation was required for proper targeting of SAMM50 to mitochondria. Thus, the strategy used in this study is useful for the identification of physiologically important human N-myristoylated proteins from human cDNA resources.     

Molecular Cloning of the Antagonist-Binding Subunit of the Glycine Receptor

Abstract

The postsynaptic receptor for the inhibitory neurotransmitter glycine is a heterooligomeric membrane protein which, after affinity purification on an antagonist column, contains three polypeptides of 48K, 58K and 93K. Sequencing of cDNA clones of the antagonist-binding 48K subunit revealed a structural organization similar to and significant amino acid homology with nicotinic acetylcholine receptor proteins. Our data suggest the existence of a set of related genes encoding transmembrane channel-forming neurotransmitter receptor polypeptides of excitable membranes.     

Identification of barley and wheat cDNA clones related to the high-Mr polypeptides of wheat gluten

We have identified 3 cDNA clones related to the high-M_r group of storage proteins in barley endosperm, the D-hordeins. A cDNA library has been constructed from wheat endosperm poly(A⁺)-RNA and screened using one of the D-hordein cDNA clones. Two wheat clones which cross-hybridised to the barley clone have been identified, by hybrid-release translation and nucleotide sequence analysis, as partial copies of mRNAs encoding the high-M_r gluten polypeptides of wheat.     

Molecular Cloning and Expression of α-Globin and β-Globin Genes from Crocodile (Crocodylus siamensis)

The first report of complete nucleotide sequences for α- and β-globin chains from the Siamese hemoglobin (Crocodylus siamensis) is given in this study. The cDNAs encoding α- and β-globins were cloned by RT-PCR using the degenerate primers and by the rapid amplification of cDNA ends method. The full-length α-globin cDNA contains an open reading frame of 423 nucleotides encoding 141 amino acid residues, whereas the β-globin cDNA contains an open reading frame of 438 nucleotides encoding 146 amino acid residues. The authenticity of both α- and β-globin cDNA clones were also confirmed by the heterologous expression in Escherichia coli (E. coli). This is the first time that the recombinant C. siamensis globins were produced in prokaryotic system. Additionally, the heme group was inserted into the recombinant proteins and purified heme-bound proteins were performed by affinity chromatography using Co²⁺-charged Talon resins. The heme-bound proteins appeared to have a maximum absorbance at 415 nm, indicated that the recombinant proteins bound to oxygen and formed active oxyhemoglobin (HbO₂). The results indicated that recombinant C. siamensis globins were successfully expressed in prokaryotic system and possessed an activity as ligand binding protein.     

Cloning of the cDNAs coding for cat growth hormone and prolactin

Characterization of the prolactin (PRL) amino acid (aa) or cDNA sequences has not been reported for any member of the Felidae family. We cloned cat growth hormone (cGH) and cat PRL (cPRL) cDNA sequences from a feline pituitary cDNA library. High homology between species allowed bovine PRL (bPRL) and bGH cDNA clones to be used to identify clones encoding the 229-aa cPRL and 216-aa cGH sequences. The cGH protein is most homologous to pig and dog GH. Similarly, cPRL shares the most aa identity to pig PRL (pPRL). Northern blot analysis revealed the mRNA size for cGH and cPRL to be approx. 1 and 1.1 kb, respectively. These results reveal that GH and PRL from the Felidae family are highly conserved to other families of GH and PRL.     

Expression and interaction of the CBLs and CIPKs from immature seeds of kidney bean (Phaseolus vulgaris L.)

Protein phosphorylation plays a key regulatory role in a variety of cellular processes. To better understand the function of protein phosphorylation in seed maturation, a PCR-based cloning method was employed and five cDNA clones (pvcipk1-5) for protein kinases were isolated from a cDNA library prepared from immature seeds of kidney bean (Phaseolus vulgaris L.). The deduced amino acid sequences showed that the five protein kinases (PvCIPK1-5) are members of the sucrose non-fermenting 1-related protein kinase type 3 (SnRK3) family, which interacts with calcineurin B-like proteins (CBLs). Two cDNA clones (pvcbl1 and 2) for CBLs were further isolated from the cDNA library. The predicted primary sequences of the proteins (PvCBL1 and 2) displayed significant identity (more than 90%) with those of other plant CBLs. Semi-quantitative RT-PCR analysis showed that the isolated genes, except pvcbl1, are expressed in leaves and early maturing seeds, whereas pvcbl1 is constitutively expressed during seed development. Yeast two-hybrid assay indicated that among the five PvCIPKs, only PvCIPK1 interacts with both PvCBL1 and PvCBL2. These results suggest that calcium-dependent protein phosphorylation-signaling via CBL-CIPK complexes occurs during seed development.     

Molecular cloning of the lectin and a lectin-related protein from common Solomon's seal (Polygonatum multiflorum)

The most prominent protein ofPolygonatum multiflorum (common Solomon's seal) rhizomes has been identified as a mannose-binding lectin. Analysis of the purified lectin demonstrated that it is a tetramer of four identical subunits of 14 kDa. Molecular cloning further revealed that the lectin from this typical Liliaceae species belongs to the superfamily of monocot mannose-binding proteins. Screening of cDNA libraries constructed with RNA isolated from buds, leaves and flowers ofP. multiflorum also yielded cDNA clones encoding a protein, which contains two tandemly arranged domains with an obvious sequence homology to the mannose-binding lectins. Molecular modelling of thePolygonatum lectin and lectin-related protein indicated that the three-dimensional structure of both proteins strongly resembles that of the snowdrop lectin. In addition, this approach suggested that the presumed carbohydrate-binding sites of the lectin can accommodate a mannose residue whereas most of the carbohydratebinding sites of the lectin-related protein cannot.Abbreviations GNA Galanthus nivalis agglutinin - HCA hydrophobic cluster analysis - LECPMA cDNA clone encoding PMA - PM30 30 kDa protein isolated fromPolygonatum multiforum - PMA Polygonatum multiflorum agglutinin - PMLRP Polygonatum multiflorum lectin-related protein     

Structure, expression, and heterogeneity of the rice seed prolamines

By screening two rice (Oryza sativa L.) seed cDNA libraries, recombinant cDNA clones encoding the rice prolamine seed storage protein were isolated. Based on cross-hybridization and restriction enzyme map analyses, these clones can be divided into two homology classes. All clones contain a single open reading frame encoding a putative rice prolamine precursor (molecular weight = 17,200) possessing a typical 14-amino acid signal peptide. The deduced primary structures of both types of prolamine polypeptides are devoid of repetitive sequences, a feature prevalent in other cereal prolamines. Clones of these two homology classes diverge mainly by insertions/deletions of short nucleotide stretches and point mutations. An isolated genomic clone about 15.5 kilobases in length displays a highly conserved 2.5-kilobase EcoRI fragment, repeated in tandem four times, each containing the prolamine coding sequence. Close homology is exhibited by the coding segments of the genomic and cDNA sequences, although the 5′ ends of the untranslated regions are widely divergent. The sequence heterogeneity displayed by these genomic and cDNA clones and large gene copy number (~80-100 copies/haploid genome) indicate that the rice prolamines are encoded by a complex multigene family.