Cloning and characterization of a novel zinc finger protein that associates with nuclear matrix

We have recently reported that the nuclei of B16 melanoma cells are intensely stained with anti-rat vitronectin (Vn) antibody, which reacts with both mouse and rat Vn. In the present study, we characterized the protein immunoreactive with the antibody using NIH3T3 cells, whose nuclei were also stained with the antibody. Western blot analysis showed that a protein with an approximate molecular weight of 75 kDa (p75), which was distinct from Vn, existed in the nuclear fraction, and, more specifically, in the nuclear matrix fraction, of NIH3T3 cells. Screening of an NIH3T3 cDNA library resulted in the isolation of a nearly full-length cDNA clone encoding p75. A database search revealed that the cDNA represents a novel gene. The deduced amino acid sequence showed that the protein is 580 amino acids long and contains two C2H2-type zinc finger motifs and glutamic acid-rich domains in the C-terminal region. When a fusion protein of green fluorescence protein and p75 was expressed in NIH3T3 cells, fluorescence was preferentially observed in the nuclei, demonstrating that the protein has a nuclear localization signal. The p75 protein, termed ZAN75, exhibited DNA-binding activity in a zinc-dependent manner. Southern blot analysis demonstrated that the ZAN75 gene exists in a single copy in the mouse genome and that a closely related gene is also present in chicken, rat, and human. Northern blot analysis showed that the ZAN75 gene is ubiquitously expressed in adult mouse tissues. In the cell cycle of NIH3T3 cells, expression was low in the G0/G1 phase, increased during the G1 phase, and persisted during the S and G2/M phases, suggesting that ZAN75 plays a role in regulating cell growth.     

Molecular cloning, sequencing, and expression of the 36 kDa protein present in pars planitis. Sequence homology with yeast nucleopore complex protein

PURPOSE: Patients with active pars planitis have increased levels of a 36 kDa protein (p-36) in their circulation. The current studies were undertaken to determine the primary structure of this protein. METHODS: A degenerate oligonucleotide probe based on the amino terminal sequence of p-36 was used to identify a clone from a human spleen cDNA library. The cDNA insert was subcloned into the EcoR1 site of pUC-19, and both strands were sequenced. Southern blot analysis was used to study the genomic hybridization pattern. p-36 cDNA was subcloned in a pSG5 expression vector, and the construct was used to transfect COS-7 cells. RESULTS: The cDNA sequence contained an open reading frame of 966 base pairs encoding a protein of 322 amino acids, an untranslated region of 322 base pairs, and 2693 base pairs at the 5' and 3' ends, respectively. The deduced amino acid sequence showed 96.8% identity with the carboxy-terminal region of a yeast nucleopore complex protein, nup 100. Southern blot analysis of human genomic DNA revealed a simple hybridization pattern. Transfection of p-36 cDNA in COS-7 cells resulted in the presence of p-36 mRNA and expression of protein. CONCLUSIONS: The 36 kDa protein (p-36) detected at increased levels in the blood of patients with active pars planitis was cloned from a human spleen cDNA library. Its deduced amino acid sequence is homologous with the carboxy-terminal region of a nucleopore complex protein. Thus, we refer to this protein as nup36.     

Cloning, expression and chromosome mapping of adducin-like 70 (ADDL), a human cDNA highly homologous to human erythrocyte adducin

From a human fetal-brain cDNA library we isolated a novel human cDNA, termed human adducin-like 70 (gene symbol ADDL), whose predicted amino acid sequence showed a high degree of homology to adducins. This cDNA clone (ADDL), which contained an open reading frame of 2,022 nucleotides encoding 674 amino acids, revealed 54%, 53%, and 59% identity in predicted amino acid sequence with alpha and beta components of human adducin and rat adducin 63, respectively. Human adducin-like 70 is likely to play an important role in the skeletal organization of the cell membrane. Northern blot analysis indicated ubiquitous expression of this gene in adult human tissues. We localized the gene to chromosome bands 10q24.2-->q24.3 by fluorescence in situ hybridization (FISH).     

Nucleotide and deduced amino acid sequences of rat myosin binding protein H (MyBP-H)

The complete nucleotide sequence of the cDNA clone encoding rat skeletal muscle myosin-binding protein H (MyBP-H) was determined and amino acid sequence was deduced from the nucleotide sequence (GenBank accession number AF077338). The full-length cDNA of 1782 base pairs(bp) contains a single open reading frame of 1454 bp encoding a rat MyBP-H protein of the predicted molecular mass 52.7 kDa and includes the common consensus 'CA__TG' protein binding motif. The cDNA sequence of rat MyBP-H show 92%, 84% and 41% homology with those of mouse, human and chicken, respectively. The protein contains tandem internal motifs array (-FN III-Ig C2-FN III-Ig C2-) in the C-terminal region which resembles to the immunoglobulin superfamily C2 and fibronectin type III motifs. The amino acid sequence of the C-terminal Ig C2 was highly conserved among MyBPs family and other thick filament binding proteins, suggesting that the C-terminal Ig C2 might play an important role in its function. All proteins belonging to MyBP-H member contains 'RKPS' sequence which is assumed to be cAMP- and cGMP-dependent protein kinase A phosphorylation site. Computer analysis of the primary sequence of rat MyBP-H predicted 11 protein kinase C (PKC) phosphorylation site, 7 casein kinase II (CK2) phosphorylation site and 4 N-myristoylation site.     

Molecular cloning and characterization of rKlk10, a cDNA encoding T-kininogenase from rat submandibular gland and kidney

We have cloned and determined the nucleotide sequence of a novel kallikrein-like mRNA, designated rKlk10*, from rat submandibular gland and kidney with the aid of the polymerase chain reaction (PCR). This cDNA contains 737 base pairs comprising the sequence encoding a mature protein of 235 amino acid residues, partial zymogen peptide, and 3' noncoding sequence. Sequence comparisons showed that rKlk10 mRNA shares 87 and 88% sequence identity with rat tissue kallikrein at nucleic acid and amino acid levels, respectively. It encodes a 26,428-Da acidic protein whose derived amino acid sequence matches completely with the partial amino acid sequence of a kallikrein-like enzyme designated as T-kininogenase, K10 protein, or antigen-gamma purified from rat submandibular gland [Xiong et al. (1990) J. Biol. Chem. 265, 2822-2827; Gutman et al. (1991) Eur. J. Biochem. 784, 1-5; Berg et al. (1991) Biochem. J. 280, 19-25]. The protein encoded by rKlk10 retains the key amino acid residues determining kallikrein cleavage specificity. Northern blot analysis with an rKlk10-specific oligonucleotide probe showed that its mRNA level in the submandibular gland is decreased dramatically by administration of the beta agonist isoproterenol. Tissue-specific expression of rKlk10 was analyzed by Northern blotting and Southern blotting of PCR-amplified cDNA, which showed that rKlk10 is expressed at high levels in the submandibular gland and low levels in the kidney but not in seven other tissues including prostate, liver, heart, adrenal gland, testes, pituitary, and pancreas. rKlk10 cDNAs cloned from the kidney and submandibular gland show sequence identity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and nucleotide sequencing of the S4 genome segment of avian reovirus S1133

The sequence of RNA genome segment S4 of the avian reovirus (ARV) strain S1133 was determined. S4 RNA is 1185 base pairs long and contains one open reading frame encoding a protein of 367 amino acid residues (40.6 kDa), the similar size as the known S4 gene product (sigma NS), with a net charge of -1 at neutral pH. The S4 RNA sequence possesses a pentanucleotide sequence UCAUC at the 3'-terminus of its plus strand like in ARV S1 and S3 segments and ten segments of mammalian reovirus (MRV). The predicted amino acid sequence comparison revealed that the homology is 44.02%, 45.71%, and 42.33% for ARV sigma NS and three serotypes of MRV sigma NS, respectively. The relatively high content of alpha-helix structure in the C-terminal portion of ARV sigma NS suggests that this protein may functionally relate to MRV sigma NS. Northern blot hybridization showed that a 32P-labeled cDNA insert S4-49 from ARV S4 RNA cross-hybridized with the corresponding RNA segments of all seven strains of ARV tested.     

Carnitine biosynthesis: identification of the cDNA encoding human gamma-butyrobetaine hydroxylase

gamma-Butyrobetaine hydroxylase (EC 1.14.11.1) is the last enzyme in the biosynthetic pathway of L-carnitine and catalyzes the formation of L-carnitine from gamma-butyrobetaine, a reaction dependent on alpha-ketoglutarate, Fe2+, and oxygen. We report the purification of the protein from rat liver to apparent homogeneity, which allowed N-terminal sequencing using Edman degradation. The obtained amino acid sequence was used to screen the expressed sequence tag database and led to the identification of a human cDNA containing an open reading frame of 1161 base pairs encoding a polypeptide of 387 amino acids with a predicted molecular weight of 44.7 kDa. Heterologous expression of the open reading frame in the yeast Saccharomyces cerevisiae confirmed that the cDNA encodes the human gamma-butyrobetaine hydroxylase. Northern blot analysis showed gamma-butyrobetaine hydroxylase expression in kidney (high), liver (moderate), and brain (very low), while no expression could be detected in the other investigated tissues.     

Cytoplasmic ribosomal protein genes of the fission yeast Schizosaccharomyces pombe display a unique promoter type: a suggestion for nomenclature of cytoplasmic ribosomal proteins in databases

We identified 34 new ribosomal protein genes in the Schizosaccharomyces pombe database at the Sanger Centre coding for 30 different ribosomal proteins. All contain the Homol D-box in their promoter. We have shown that Homol D is, in this promoter type, the TATA-analogue. Many promoters contain the Homol E-box, which serves as a proximal activation sequence. Furthermore, comparative sequence analysis revealed a ribosomal protein gene encoding a protein which is the equivalent of the mammalian ribosomal protein L28. The budding yeast Saccharomyces cerevisiae has no L28 equivalent. Over the past 10 years we have isolated and characterized nine ribosomal protein (rp) genes from the fission yeast S.pombe . This endeavor yielded promoters which we have used to investigate the regulation of rp genes. Since eukaryotic ribosomal proteins are remarkably conserved and several rp genes of the budding yeast S.cerevisiae were sequenced in 1985, we probed DNA fragments encoding S.cerevisiae ribosomal proteins with genomic libraries of S.pombe . The deduced amino acid sequence of the different isolated rp genes of fission yeast share between 65 and 85% identical amino acids with their counterparts of budding yeast.     

Primary structure of Drosophila ribosomal protein L14 and identification of conserved protein motifs

Determination of the primary structure of individual ribosomal proteins is important for understanding their functions and organization within the ribosome. I have sequenced a cDNA that encodes a Drosophila homolog of the rat ribosomal protein L14. The cDNA sequence was 601 nucleotides long, with an open reading frame encoding a protein of 166 amino acids. Homology searches revealed 34-38% sequence identity to the rat and yeast L14 ribosomal proteins. There were also extensive homologies to sequences in the EST database, which are likely to encode portions of L14. Analysis of sequence comparisons revealed several highly conserved regions, one of which is related to a portion of ribosomal protein L27. The sizes of the L14 proteins vary between different species, with most of the variability confined to the C-terminal region.     

A novel glutathione peroxidase in bovine eye. Sequence analysis, mRNA level, and translation

Bovine ciliary body contains a selenium-independent glutathione peroxidase (GPX) with a molecular mass of about 100 kDa that is composed of four identical subunits and exhibits no glutathione S-transferase activity. In this study, we isolated cDNA clones and determined the nucleotide sequence to deduce the primary structure of the enzyme. The cDNA contained 672 base pairs encoding a polypeptide with an estimated molecular mass of 25,064 Da. Translation of bovine ciliary mRNA produced a protein which was immunologically indistinguishable from GPX and showed high enzyme activity. The encoded amino acid sequence of the protein was 95% identical with that of a human keratinocyte gene product expressed in response to keratinocyte growth factor. It also showed sequence identity to bacterial alkyl hydroperoxide reductases and thiol specific antioxidant enzymes. GPX mRNA level was highest in the ciliary body, followed by the retina and iris. In various rat organs, the level of GPX mRNA was highest in the lung, followed by the muscle, liver, eye, heart, testis, thymus, kidney, and spleen. A very low level of mRNA was detected in the brain. Enzyme-linked immunosorbent assay with an antibody raised against the NH2-terminal sequence of GPX detected GPX protein in all rat tissues examined.     

Molecular cloning of human G alpha q cDNA and chromosomal localization of the G alpha q gene (GNAQ) and a processed pseudogene

G alpha q is the alpha subunit of one of the heterotrimeric GTP-binding proteins that mediates stimulation of phospholipase C beta. We report the isolation and characterization of cDNA clones from a frontal cortex cDNA library encoding human G alpha q. The encoded protein is 359 amino acids long and is identical in all but one amino acid residue to mouse G alpha q. Analysis of human genomic DNA reveals an intronless sequence with strong homology to human G alpha q cDNA. In comparison to G alpha q cDNA, this genomic DNA sequence includes several small deletions and insertions that alter the reading frame, multiple single base changes, and a premature termination codon in the open reading frame, hallmarks of a processed pseudogene. Probes derived from human G alpha q cDNA sequence map to both chromosomes 2 and 9 in high-stringency genomic blot analyses of DNA from a panel of human-rodent hybrid cell lines. PCR primers that selectively amplify the pseudogene sequence generate a product only when DNA containing human chromosome 2 is used as the template, indicating that the authentic G alpha q gene (GNAQ) is located on chromosome 9. Regional localization by FISH analysis places GNAQ at 9q21 and the pseudogene at 2q14.3-q21.     

Thallium poisoning presenting with abdominal colic, paresthesia, and irritability

We have determined the complete nucleotide sequence for the cDNA encoding rat eosinophil major basic protein (MBP) using the rapid amplification of cDNA ends (RACE) procedure. The deduced amino acid sequence revealed that the rat prepro-MBP has three functional domains, namely the signal peptide, the acidic peptide that contains numerous acidic amino acids, and the mature MBP, as in human and guinea pig MBP.     

Immunogenicity and cross-reactivity with idiotypic IgA of VH CDR3 peptide in multiple myeloma

We have cloned the S6 ribosomal protein encoding gene from a Leishmania infantum cDNA library. This parasite protozoon, responsible for leishmaniasis in Europe, is able to undergo developmental changes in vitro and results a good model to study cell differentiation processes. The LiS6 protein sequence indicates its pertinence to the S6 protein family, related to the early mechanisms of cell division, differentiation and activation, and shows an intermediate position between the yeasts and higher eukaryotes. Thus, LiS6 protein has the same amino acid length as that of the higher eukaryotes and certain common features such nucleus entrance sequences and several kinase phosphorylation sites. However, the key functional protein kinase C phosphorylation sites are at different locations and present several threonine instead of the usual serine residues. The gene structural analysis suggest the presence of three different encoding genes that do not present remarkable changes along the different phases of the parasite.