Neoplastic transformation and tumorigenesis associated with overexpression of IMUP-1 and IMUP-2 genes in cultured NIH/3T3 mouse fibroblasts

Immortalization-upregulated protein 1 (IMUP-1) and immortalization-upregulated protein 2 (IMUP-2) genes have been recently cloned and are known to be involved in SV40-mediated immortalization. IMUP-1 and IMUP-2 genes were strongly expressed in various cancer cell lines and tumors, suggesting the possibility that they might be involved in tumorigenicity. To directly elucidate the functional role of IMUP-1 and IMUP-2 on neoplastic transformation and tumorigenicity, we stably transfected IMUP-1 and IMUP-2 into NIH/3T3 mouse fibroblast cells. Cellular characteristics of the neoplastic transformation were assessed by transformation foci, growth in soft agar, and tumor development in nude mice. We found that IMUP-1 and IMUP-2 overexpressing cells showed altered growth properties, anchorage-independent growth in soft agar and inducing tumor in nude mice. Furthermore, IMUP-1 and IMUP-2 transformants proliferated in reduced serum and shortened cell cycle. These results suggest that ectopic overexpression of IMUP-1 and IMUP-2 may play an important role in acquiring a transformed phenotype, tumorigenicity in vivo, and be related to cellular proliferation.     

Two MAR DNA-binding proteins of the pea nuclear matrix identify a new class of DNA-binding proteins

Four MAR-binding proteins of 60, 65, 70 and 72 kDa have been detected by South-Western blotting and isolated from pea nuclear matrices. Two cDNAs encoding the 60 and 65 kDa proteins (MARBP-1 and MARBP-2) were isolated from a pea leaf cDNA library by screening with a PCR product obtained using degenerate primers based on an amino acid sequence from the 60 kDa protein. The proteins of 560 and 550 amino acids are 86% identical and contain several KKD/E repeats near the C-terminus. Escherichia coli-expressed MARBP-1 specifically binds A/T-rich MAR DNA. The interaction of MARBP-1/MARBP-2 with MAR DNA involves novel DNA-binding motifs. The MARBP-1 and MARBP-2 genes are expressed in a range of pea tissues and are encoded by genes at different loci. MARBP-1 and MARBP-2 are homologous to yeast nucleolar proteins Nop56p and Nop58p, which are involved in ribosome biogenesis, and to similar highly conserved proteins in other eukaryotes and in archaebacteria. MARBP-1 and MARBP-2 may have multifunctional roles in chromatin organisation and ribosome biogenesis.     

NARG2 encodes a novel nuclear protein with (S/T)PXX motifs that is expressed during development.

We previously identified a partial expressed sequence tag clone corresponding to NARG2 in a screen for genes that are expressed in developing neurons and misexpressed in transgenic mice that lack functional N-methyl-d-aspartate receptors. Here we report the first characterization of the mouse and human NARG2 genes, cDNAs and the proteins that they encode. Mouse and human NARG2 consist of 988 and 982 amino acids, respectively, and share 74% identity. NARG2 does not display significant homology to other known genes, and lower organisms such as Saccharomyces cerevisiae, Drosophila melanogaster and Fugu rubripes appear to lack NARG2 orthologs. In vitro translation of the mouse cDNA yields a 150 kDa protein. NARG2 localizes to the nucleus in transfected cells, and deletion of a canonical basic nuclear localization signal suggests that this and other sequences in the protein cooperate for nuclear targeting. NARG2 consists of 16 exons in both mice and humans, 11 of which are identical in length, and alternative splicing is evident in both species. Exon 10 is the largest, and exhibits a much higher rate of nonsynonymous nucleotide substitution than the others. In addition, NARG2 contains (S/T)PXX motifs (11 in mouse NARG2, six in human NARG2). Northern blot analysis and RNase protection demonstrated that NARG2 is expressed at relatively high levels in dividing and immature cells, and that it is down-regulated upon terminal differentiation. The results indicate that NARG2 encodes a novel (S/T)PXX motif-containing nuclear protein, and suggest that NARG2 may play an important role in the early development of a number of different cell types.     

Characterization of cDNA clones encoding a human fibroblast caldesmon isoform and analysis of caldesmon expression in normal and transformed cells

Overlapping cDNA clones encoding a low M gamma human nonmuscle caldesmon isoform (HUM 1-CaD) span the entire coding region (538 amino acids) as well as 111 base pairs (bp) of 5'-noncoding and 1249 bp of 3'-noncoding region. Northern blot probes derived from either the coding or 3'-noncoding region hybridized to a 4.3-kilobase mRNA in nonmuscle cells and a 5.2-kilobase mRNA in stomach tissue. Primer extension results indicated that the 5'-noncoding region of the HUM 1-CaD mRNA is approximately 700 bp in length and also suggested that 1-CaD mRNAs with common 5'-noncoding regions are expressed in both liver and fibroblast cells. Comparisons of the human, rat, and chicken 1-CaD amino acids sequences demonstrated that although each isoform has unique characteristics, extensive regions of conservation exist. Amino acids 27-53 and 97-127 are 100% identical in these isoforms while amino acids 297-531 of HUM 1-CaD are 94 and 85% identical to the rat and chicken 1-CaDs, respectively. In addition, the levels of HUM 1-CaD mRNA and protein appeared to be decreased by 2-4 fold in the transformed derivatives of KD and WI38 cell lines as judged by Northern and Western blot analysis. The results suggest that the decrease of 1-CaD protein in these transformed cells is a direct result of decreased 1-CaD mRNA synthesis and/or increased mRNA turnover.     

Molecular cloning and expression of prohormone convertases PC1 and PC2 in the pituitary gland of the bullfrog, Rana catesbeiana

We cloned cDNAs encoding PC1 and PC2 from a cDNA library constructed for the anterior pituitary gland of the bullfrog (Rana catesbeiana) and sequenced them. The bullfrog PC1 cDNA consisted of 2972 base pairs (bp) with an open reading frame of 2208 bp and encoded a protein of 736 amino acids, including a putative signal peptide of 26 amino acids. The protein showed a high homology to R. ridibunda PC1 (95.1%) and mammalian PC1 (72.6%). The bullfrog PC2 cDNA consisted of 2242 bp with an open reading frame of 1914 bp and encoded a protein of 638 amino acids, including a putative signal peptide of 23 amino acids. This protein showed a high homology to R. ridibunda PC2 (95.5%) and mammalian PC2 (84.8%). The catalytic triad of serine proteinases of the subtilisin family was found at Asp-168, His-209, and Ser-383 in the PC1 protein and at Asp-167, His-208, and Ser-384 in the PC2 protein. In situ hybridization staining revealed that PC2 mRNA was detected in corticotrope cells of the tadpoles, but not in those of the adults. In the adult, only PC1 mRNA was detected in the pars distalis but both PC1 and PC2 mRNAs were detected in the pars intermedia. The data also showed that PC1 mRNA was expressed in gonadotrope cells.     

Peroxisomal acetoacetyl-CoA thiolase of an n-alkane-utilizing yeast, Candida tropicalis.

Two genes encoding acetoacetyl-CoA thiolase (thiolase I; EC 2.3.1.9), whose localization in peroxisomes was first found with an n-alkane-utilizing yeast, Candida tropicalis, were isolated from the lambda EMBL3 genomic DNA library prepared from the yeast genomic DNA. Nucleotide sequence analysis revealed that both genes contained open reading frames of 1209 bp corresponding to 403 amino acid residues with methionine at the N-terminus, which were named as thiolase IA and thiolase IB. The calculated molecular masses were 41,898 Da for thiolase IA and 41,930 Da for thiolase IB. These values were in good agreement with the subunit mass of the enzyme purified from yeast peroxisomes (41 kDa). There was an extremely high similarity between these two genes (96% of nucleotides in the coding regions and 98% of amino acids deduced). From the amino acid sequence analysis of the purified peroxisomal enzyme, it was shown that thiolase IA and thiolase IB were expressed in peroxisomes at an almost equal level. Both showed similarity to other thiolases, especially to Saccharomyces uvarum cytosolic acetoacetyl-CoA thiolase (65% amino acids of thiolase IA and 64% of thiolase IB were identical with this thiolase). Considering the evolution of thiolases, the C. tropicalis thiolases and S. uvarum cytosolic acetoacetyl-CoA thiolase are supposed to have a common origin. It was noticeable that the carboxyl-terminal regions of thiolases IA and IB contained a putative peroxisomal targeting signal, -Ala-Lys-Leu-COOH, unlike those of other thiolases reported hitherto.     

Cloning, purification and characterization of two components of phenol hydroxylase from Rhodococcus erythropolis UPV-1

Phenol hydroxylase that catalyzes the conversion of phenol to catechol in Rhodococcus erythropolis UPV-1 was identified as a two-component flavin-dependent monooxygenase. The two proteins are encoded by the genes pheA1 and pheA2, located very closely in the genome. The sequenced pheA1 gene was composed of 1,629 bp encoding a protein of 542 amino acids, whereas the pheA2 gene consisted of 570 bp encoding a protein of 189 amino acids. The deduced amino acid sequences of both genes showed high homology with several two-component aromatic hydroxylases. The genes were cloned separately in cells of Escherichia coli M15 as hexahistidine-tagged proteins, and the recombinant proteins His₆PheA1 and His₆PheA2 were purified and its catalytic activity characterized. His₆PheA1 exists as a homotetramer of four identical subunits of 62 kDa that has no phenol hydroxylase activity on its own. His₆PheA2 is a homodimeric flavin reductase, consisting of two identical subunits of 22 kDa, that uses NAD(P)H in order to reduce flavin adenine dinucleotide (FAD), according to a random sequential kinetic mechanism. The reductase activity was strongly inhibited by thiol-blocking reagents. The hydroxylation of phenol in vitro requires the presence of both His₆PheA1 and His₆PheA2 components, in addition to NADH and FAD, but the physical interaction between the proteins is not necessary for the reaction.