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Chicken insulin-like growth factor binding protein (IGFBP)-5: conservation of IGFBP-5 structure and expression during evolution.

A complementary DNA (cDNA) clone encoding chicken insulin-like growth factor binding protein-5 (cIGFBP-5) was isolated. The nucleotide sequence of the 1236-bp clone encodes a mature polypeptide of 251 amino acids with a calculated molecular weight of approximately 28.2 kDa. The deduced amino acid sequence is 83% identical to human IGFBP-5. Labeled cIGFBP-5 cDNA detected a single mRNA transcript of approximately 6 kb by Northern blot analysis of various tissues obtained from embryonic and 6 weeks post-hatch chickens; interestingly, adult heart showed an approximately 10-fold increase in cIGFBP-5 mRNA relative to embryonic heart. The pattern of IGFBP-5 mRNA expression in chicken tissues was similar to that found in mammals during fetal and extrauterine life. In addition, IGFBP-5 mRNA was abundant in primary cultures of chicken myoblasts throughout in vitro differentiation and fusion. The conservation of IGFBP-5 primary structure and expression pattern across vertebrate species suggests conservation of important functions during evolution, particularly in muscle differentiation.

Amino Acid Sequence↗

Genetic basis of hemolytic anemia caused by pyrimidine 5' nucleotidase deficiency.

Pyrimidine 5' nucleotidase (P5'N-1) deficiency is an autosomal recessive condition causing hemolytic anemia characterized by marked basophilic stippling and the accumulation of high concentrations of pyrimidine nucleotides within the erythrocyte. It is implicated in the anemia of lead poisoning and is possibly associated with learning difficulties. Recently, a protein with P5'N-1 activity was analyzed and a provisional complementary DNA (cDNA) sequence published. This sequence was used to study 3 families with P5'N-1 deficiency. This approach generated a genomic DNA sequence that was used to search GenBank and identify the gene for P5'N-1. It is found on chromosome 7, consists of 10 exons with alternative splicing of exon 2, and produces proteins 286 and 297 amino acids long. Three homozygous mutations were identified in this gene in 4 subjects with P5'N-1 deficiency: codon 98 GAT-->GTT, Asp-->Val (linked to a silent polymorphism codon 92, TAC-->TAT), codon 177, CAA-->TAA, Gln-->termination, and IVS9-1, G-->T. The latter mutation results in the loss of exon 9 (201 bp) from the cDNA. None of these mutations was found in 100 normal controls. The DNA analysis was complicated by P5'N-1 pseudogenes found on chromosomes 4 and 7. This study is the first description of the structure and location of the P5'N-1 gene, and 3 mutations have been identified in affected patients from separate kindreds. (Blood. 2001;97:3327-3332)

5'-Nucleotidase↗

Primary structure and functional expression of a novel non-selective cation channel.

A non-selective cation channel is believed to play important roles in various tissues. A novel complementary DNA encoding non-selective cation channel was isolated from MIN6, a mouse insulin secreting beta-cell line. This channel (mNSC1) conducts predominantly monovalent cations in Xenopus oocytes and is selective for cations over anions (PK/PCl = 10). The current was completely blocked by lanthanum and niflumate. The mNSC1 of 423 amino acids contains a characteristic leucine repeat and unique membrane topology. The messenger RNA of this channel are abundant in the brain, heart, and lung. We may therefore conclude that studies with this channel will provide important information for understanding physiological functions of the excitable cells as well as non-excitable epithelia.

Amino Acid Sequence↗

Constitutive androstane receptor (CAR) as a potential sensing biomarker of persistent organic pollutants (POPs) in aquatic mammal: molecular characterization, expression level, and ligand profiling in Baikal seal (Pusa sibirica).

To characterize the function of constitutive active/androstane receptor (CAR) in aquatic mammals, CAR complementary DNA (cDNA) was cloned from the liver of Baikal seal (Pusa sibirica) from Lake Baikal, Russia, and the messenger RNA (mRNA) expression levels in various tissues/organs of the wild population and the CAR ligand profiles were investigated. The seal CAR cDNA had an open reading frame of 1047 bp encoding 348 amino acids that revealed 74-84% amino acid identities with CARs from rodents and human. The mRNA expression profile of tissues/organs represented that Baikal seal CAR was predominantly expressed in the liver followed by heart and intestine. The expression analysis of hepatic CAR mRNA showed no correlation with expression of cytochrome P450 (CYP) 1A, 1B, 2B, 2C, and 3A-like proteins, indicating that the CAR expression level may not be the sole determinant of the regulation of these CYP expressions in the seal liver. There was no significant correlation between CAR expression and any of the persistent organic pollutants (POPs) levels. Furthermore, we performed an in vitro CAR transactivation assay using MCF-7 cells transfected with Baikal seal CAR expression plasmid and (NR1)(3)-luciferase reporter gene plasmid. In the transactivation analysis of Baikal seal CAR, neither repression by androstanol and androstenol, nor activation by estrone and estradiol, which are recognized as endogenous ligands for mouse and human CARs, was detected. On the other hand, bile acids such as chenodeoxycholic acid, deoxycholic acid, and lithocholic acid activated the seal CAR as well as mouse CAR. As for exogenous chemicals, the seal CAR was transactivated by a human CAR agonist, 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime), but not by a mouse CAR agonist, (1,4-bis[2-(3,5-dichloropyridyloxy)]benzene). In addition, the seal CAR was also activated by polychlorinated biphenyls (PCBs) (Kanechlor-500, International Union of Pure and Applied Chemistry No. PCB153; 2,2',4,4',5,5'-hexachlorobiphenyl and PCB180; 2,2',3,4,4',5,5'-heptachlorobiphenyl), and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (p,p'-DDT) and its metabolite, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE). The seal CAR responded more sensitively to PCBs than the mouse CAR. Based on the results of CAR transactivation assay, the lowest observable effect levels of Kanechlor-500, PCB153, PCB180, p,p'-DDT, and p,p'-DDE in Baikal seal were estimated to be 10, 20, 20, 10, and 10 ppm on wet weight basis, respectively. These results suggest that CAR is conserved in diverse mammalian species including seals. Whereas the seal CAR-mediated gene transcription may potentially be a sensitive response to the exposure of certain POPs, the ligand profile of seal CAR may be different from those of other mammalian CARs. This study indicates that CAR-mediated responses may be useful information to assess the ecotoxicological risk of xenobiotics such as POPs in wildlife but the previous results derived from rodent and human CAR may not be applicable to the risk assessment in wild species.

Amino Acid Sequence↗

Strand-selective transcription of globin genes in rabbit erythroid cells and chromatin.

In order to investigate the symmetry of globin gene transcription, complementary RNA (cRNA) was synthesized using rabbit globin complementary DNA (cDNA) as a template for Escherichia coli DNA-dependent RNA polymerase (RNA nucleotidyltransferase). The cRNA hybridized specifically to its own cDNA template but not to sheep cDNA, rabbit globin mRNA, or poly(dT). Hybridization studies with cRNA demonstrated that RNA sequences transcribed from the DNA strand complementary to the globin gene region (anti-strand) were not present in cellular, total nuclear, or fractionated nuclear RNA from rabbit marrow. Such sequences were detected in RNA transcribed from rabbit marrow chromatin by E. coli or sheep liver RNA polymerases, but amounted to less than 50% of the globin mRNA sequences present in the same transcript. The evidence indicates that globin mRNA transcription is predominantly DNA strand specific.

Animals↗

Molecular cloning of the mouse Ltbp-1 gene reveals tissue specific expression of alternatively spliced forms.

Latent transforming growth factor binding proteins (Ltbp-1, -2, -3 and -4) and fibrillins (Fbn-1 and -2) are structurally related cysteine-rich extracellular matrix proteins that localize to the 10 nm microfibrils. Ltbp-1 is thought to promote the secretion and proper folding of the small latent transforming growth factor beta (TGF-beta) complex (TGF-beta plus its propeptide) and is implicated in sequestering it in the extracellular matrix. Here we report the isolation of the mouse Ltbp-1 complementary DNA (cDNA) and gene. The longer form of the Ltbp-1 cDNA encodes a predicted 1713 amino acid protein containing 18 epidermal growth factor-like repeats, four 8-cysteine domains and several motifs that suggest interactions with alpha(IV)beta(1) and alpha(9)beta(1) integrins. Northern blotting analyses indicate that long and short Ltbp-1 transcripts are widely expressed in adult mouse tissues and most abundantly expressed in heart. Ltbp-1 is a single copy gene that maps to chromosome 17, band E (1-3) and encompasses more than 212 kb. The Ltbp-1 gene contains 34 exons and shows a similar organization to the LTBP-2 gene, suggesting that these genes originated from a common ancestral gene.

Alternative Splicing↗

A novel missense mutation (G2320R) in thyroglobulin causes hypothyroidism in rdw rats.

The rdw rat is a hereditary hypothyroid variant initially derived from the Wistar-Imamichi strain. Proteome analysis by two-dimensional gelelectrophoresis showed that molecular chaperones accumulated in the thyroid glands, suggesting retention of abnormal proteins in the endoplasmic reticulum (ER). Anatomical studies indicated that thyroglobulin (Tg) was not secreted into the follicular lumina, but retained in the dilated ER. Sequencing of the entire Tg complementary DNA from the rdw rat revealed a missense mutation (G2320R) in the acetylcholinesterase-like domain at the 2320th amino acid residue. Carbohydrate residues of the G2320R Tg mutant were of the high-mannose ER type, as shown by sensitivity to the treatment with endoglycosidase H. Molecular chaperones, GRP94, GRP78, and calreticulin, were all accumulated in the rdw rat thyroid glands. Computer analysis of protein secondary structure predicted that the mutation would cause extension of the helix where beta-sheet and turns were formed in the normal Tg. Altered folding of Tg might account for the impaired intracellular transport of Tg and activated premature degradation by the same mechanism as in ER storage diseases.

Amino Acid Sequence↗

Initial assessment of human gene diversity and expression patterns based upon 83 million nucleotides of cDNA sequence.

In an effort to identify new genes and analyse their expression patterns, 174,472 partial complementary DNA sequences (expressed sequence tags (ESTs)), totalling more than 52 million nucleotides of human DNA sequence, have been generated from 300 cDNA libraries constructed from 37 distinct organs and tissues. These ESTs have been combined with an additional 118,406 ESTs from the database dbEST, for a total of 83 million nucleotides, and treated as a shotgun sequence assembly project. The assembly process yielded 29,599 distinct tentative human consensus (THC) sequences and 58,384 non-overlapping ESTs. Of these 87,983 distinct sequences, 10,214 further characterize previously known genes based on statistically significant similarity to sequences in the available databases; the remainder identify previously unknown genes. Thirty tissues were sampled by over 1,000 ESTs each; only eight genes were matched by ESTs from all 30 tissues, and 227 genes were represented in 20 or more of the tissues sampled with more than 1,000 ESTs. Approximately 40% of identified human genes appear to be associated with basic energy metabolism, cell structure, homeostasis and cell division, 22% with RNA and protein synthesis and processing, and 12% with cell signalling and communication.

Adult↗

Molecular cloning and characterization of an adaptor protein Shc isoform from Xenopus laevis oocytes.

In order to gain further insight into IGF-1 receptor signaling in Xenopus laevis oocytes and embryos, we have undertaken the characterization of the adapter protein Shc and studied its implication in oocyte maturation induced after IGF-1 receptor activation, especially since expression of this molecule has been indirectly evidenced in Xenopus oocytes, eggs and embryos. We report herein the cloning from Xenopus postvitellogenic oocytes of a complementary DNA encoding a protein of 470 amino acids which shows the higher identity with the mammalian adaptor protein p52(ShcA). Western blot analysis using homologous antibodies evidenced a 60-kDa protein, p60(Xl)(Shc), that is predominantly expressed in oocytes and in early embryos. We also demonstrate that, like p60(Xl)(Shc), Grb2 and the guanine nucleotide exchange factor Sos are expressed in oocytes throughout vitellogenesis and in early embryos and that overexpression of a dominant-negative form of Grb2 specifically inhibits insulin-induced resumption of meiosis. We finally show that Grb2 binds to p60(Shc) in oocytes specifically upon insulin treatment. Altogether, these results suggest that Shc and Grb2-Sos are implicated in ras-dependent Xenopus oocyte maturation induced by insulin/IGF-1; they also indicate that inability of insulin/IGF-1 to activate the Ras-MAPK cascade in vitellogenic oocytes does not result from an insufficient expression level of Shc, Grb2 and Sos.

Adaptor Proteins, Signal Transducing↗

Diurnal change of cucumber CPD photolyase gene (CsPHR) expression and its physiological role in growth under UV-B irradiation.

Complementary DNA encoding a putative cyclobutane pyrimidine dimer (CPD)-specific DNA photolyase (CPD photolyase) was isolated from cucumber leaves. The deduced amino acid sequence of the cDNA exhibited high similarity to that of the Arabidopsis CPD photolyase. Transformation with the cDNA restored the impaired photorepair activity of an Escherichia coli mutant, indicating that this cDNA encodes a functional cucumber CPD photolyase (CsPHR). The level of CsPHR transcripts estimated by quantitative RT-PCR as well as the CPD photorepair activity in the cucumber first leaves showed diurnal changes, peaking at 09 : 00 and 12 : 00, respectively. Supplemental UV-B irradiation in the middle of the light period had little effect on the growth of the first leaves, while the supplemental irradiation in the early morning or late afternoon strongly retarded the leaf growth. These results suggest that the diurnal change in CPD photorepair activity, which is presumably regulated by the transcript level of CsPHR, may play an important role in minimizing the growth inhibition due to UV-B irradiation.

Amino Acid Sequence↗

Rat zinc-fingers and homeoboxes 1 (ZHX1), a nuclear factor-YA-interacting nuclear protein, forms a homodimer.

Zinc-fingers and homeoboxes 1 (ZHX1) is a protein which interacts with the activation domain of the A subunit of nuclear factor-Y. To analyze the physiological role(s) of ZHX1, we searched ZHX1-interacting protein(s) using a yeast two-hybrid system. The rat counterpart of ZHX1 cDNAs was cloned from an ovarian granulosa cell complementary DNA (cDNA) library, indicating that ZHX1 is able to form a homodimer. An analysis of the nucleotide sequence and its deduced amino acid sequence show that rat ZHX1 consists of 873 amino acid residues. Northern blot analysis shows that ZHX1 messenger RNA is expressed ubiquitously and that the level in the ovary are not regulated by gonadotropins. Furthermore, transfection experiments with green fluorescence protein (GFP) expression vectors into human embryonic kidney HEK293 cells reveal that full-length ZHX1 fused to the GFP is localized in the nuclei. Thus, we report on the molecular cloning, expression and characterization of full-length rat ZHX1 cDNA.

Amino Acid Sequence↗

Structure and chromosomal assignment of the sterol 12alpha-hydroxylase gene (CYP8B1) in human and mouse: eukaryotic cytochrome P-450 gene devoid of introns.

Sterol 12alpha-hydroxylase (CYP8B1) is a hepatic cytochrome P-450 that controls the ratio of cholic acid over chenodeoxycholic acid in bile and thus controls the solubility of cholesterol. Both the human and the mouse CYP8B1 complementary DNA and gene were cloned and structurally characterized. Surprisingly, the genomic DNA from both species was found to lack introns. The major transcript of the human gene was estimated to be 3950 bp, and the putative promoter region was estimated to be at least 1360 bp. The murine structural gene was found to span approximately 3 kb. By using FISH and radiation hybrid mapping techniques, the human CYP8B1 gene was located to chromosome 3p21.3-p22, whereas FISH mapped the murine counterpart to chromosome 9qF4, a region that is homologous to the third human chromosome. The results from the chromosome mapping and Southern blotting indicated that the gene is present in a single copy. Transcription of the mouse and human CYP8B1 genes was initiated from a position situated 51 and 35 bases, respectively, downstream of a consensus TATA box. A homology of 21% for the promoter regions of mouse and human may indicate differences in transcriptional regulation. Although a potent induction of CYP8B1 mRNA was observed upon starvation of mice, the mechanism behind this effect was not revealed by analysis of the promoter for potential cis-acting elements. In the human promoter, several possible cis-acting regions were identified but none of them could be directly related to bile acid metabolism. After transfection of COS cells with the human coding region, mRNA and enzymatic activity for the 12alpha-hydroxylase were identified. This is the first mammalian cytochrome P-450 gene reported to lack introns. The importance of this structural feature for evolution and gene regulation is discussed.

Amino Acid Sequence↗

Identification and structural analysis of human RBM8A and RBM8B: two highly conserved RNA-binding motif proteins that interact with OVCA1, a candidate tumor suppressor.

The OVCA1 gene is a candidate for the breast and ovarian tumor suppressor gene at chromosome 17p13.3. To help determine the function(s) of OVCA1, we used a yeast two-hybrid screening approach to identify OVCA1-associating proteins. One such protein, which we initially referred to as BOV-1 (binder of OVCA1-1) is 173 or 174 amino acids in length and appears to be a new member of a highly conserved RNA-binding motif (RBM) protein family that is highly conserved evolutionarily. Northern blot analysis revealed that BOV-1 is ubiquitously expressed and that three distinct messenger RNA species are expressed, 1-, 3.2-, and 5.8-kb transcripts. The 1-kb transcript is the most abundant and is expressed at high levels in the testis, heart, placenta, spleen, thymus, and lymphocytes. Using fluorescence in situ hybridization and the 5.8-kb complementary DNA probe, we determined that BOV-1 maps to both chromosome 5q13-q14 and chromosome 14q22-q23. Further sequence analysis determined that the gene coding the 1- and the 3.2-kb transcripts (HGMW-approved gene symbol RBM8A) maps to 14q22-q23, whereas a second highly related gene coding for the 5.8-kb transcript resides at chromosome 5q13-q14 (HGMW-approved gene symbol RBM8B). The predicted proteins encoded by RBM8A and RBM8B are identical except that RBM8B is 16 amino acids shorter at its N-terminus. Molecular modeling of the RNA-binding domain of RBM8A and RBM8B, based on homology to the sex-lethal protein of Drosophila, identifies conserved residues in the RBM8 protein family that are likely to contact RNA in a protein-RNA complex. The conservation of sequence and structure through such an evolutionarily divergent group of organisms suggests an important function for the RBM8 family of proteins.

Amino Acid Motifs↗

Characterization of the nm23-M2, nm23-M3 and nm23-M4 mouse genes: comparison with their human orthologs.

The nm23 gene family is thought to be involved in physiopathological processes such as growth, differentiation and cancer promotion, progression or metastasis. We report here the mouse nm23-M3 and nm23-M4 complementary DNA sequences and the genomic cloning, characterization and tissue expression pattern of the nm23-M2, nm23-M3 and nm23-M4 genes, in comparison with their human and rat orthologs and with the human nm23-H1 and mouse nm23-M1 genes. The organization and structure of the members of this gene family are remarkably similar in human and rodents. Accordingly, the striking similarities between the human and mouse nm23 genes enable the use of mouse transgenic and knock-out models for studying the role of nucleoside diphosphate kinase isoforms in human physiopathology.

Amino Acid Sequence↗

Differential expression of C-protein isoforms in developing and degenerating mouse striated muscles.

With the aim of clarifying the roles of C-protein isoforms in developing mammalian skeletal muscle, we cloned the complementary DNA (cDNAs) encoding mouse fast (F) and slow (S) skeletal muscle C-proteins and determined their entire sequences. Northern blotting with these cDNAs together with mouse cardiac (C) C-protein cDNA was performed. It revealed that in adult mice, C, F, and S isoforms are expressed in a tissue-specific fashion, although the messages for both F and S isoforms are transcribed in extensor digitorum longus muscle, which has been categorized as a fast muscle. In addition, although C isoform is expressed first and transiently during development of chicken skeletal muscles, C isoform is not expressed in mouse skeletal muscles at all through the developmental stages; S isoform is first expressed, followed by the appearance of F isoform. Finally, in dystrophic mouse skeletal muscles, the expression of S isoform is increased as it is in dystrophic chicken muscle. These observations suggest that mutations in C isoform (MyBP-C) do not lead to any disturbance in skeletal muscle, although they may lead to familial hypertrophic cardiomyopathy. We also suggest that the expression of S isoform may be stimulated in degenerating human dystrophic muscles.

Amino Acid Sequence↗

Differences in the abundance of variably spliced transcripts for the second asialoglycoprotein receptor polypeptide, H2, in normal and transformed human liver.

The human hepatic asialoglycoprotein receptor comprises two homologous polypeptides designated H1 and H2. Two distinct complementary DNA clones encoding these receptor subunits have been previously isolated from the human hepatoblastoma cell line HepG2. We discovered that multiple variants of H2 transcripts exist both in HepG2 cells and in the normal human liver that, at least in part, appear to be the result of alternative splicing events. We have found that (a) the complementary DNA clone for H2 previously isolated from HepG2 cells, characterized by a 57-nucleotide insertion within the 5' end of the complementary DNA that is absent from H1, represented only one third of H2-related sequences in an unamplified normal human liver complementary DNA library and less than 10% of H2 clones in HepG2 cells; (b) the predominant message for H2 expressed in the liver and HepG2 cells, designated L-H2, appeared to represent the fully processed product of the gene encoding both L-H2 and H2; and (c) a variant H2 transcript existed in HepG2 cells, designated H2', that contained a novel, 5' 88-bp nucleotide insertion. Poly(A+) RNA analysis of the normal liver and HepG2 cells by complementary RNA hybridization and ribonuclease protection corroborated the observations made during the screening of complementary DNA libraries regarding the abundance of the various messages. A striking incongruity was found between the levels of messenger RNA containing the H2-specific 57-nucleotide sequence and the levels of polypeptide expressed in the liver and HepG2 cells as recognized by antiserum specifically raised against this sequence.

Asialoglycoprotein Receptor↗

Cloning, sequence analysis, and processing of the rat and human atrial natriuretic peptide precursors.

Complementary DNA sequences and structural genes encoding the atrial natriuretic peptide precursor (prepro-ANP) have been cloned. Analysis of DNA sequences, complementary to rat atrial prepro-ANP mRNA, has revealed that the various natriuretic peptides isolated from rat atrium reside at the carboxy terminus of a 152-amino-acid precursor protein. The human gene, comprised of three exons and two intervening sequences, encodes a protein of 151 amino acids highly homologous to the rat precursor. Although putative proteolytic processing sites can be identified throughout the prepro-ANP amino acid sequence, the natural form of the mature ANP has not been identified. Therefore, the sites and mechanisms of prepro-ANP processing to mature peptides forms are unknown. However, the successful cloning of the prepro-ANP gene and corresponding cDNAs provide the necessary molecular tools to address these fundamental questions relating to the regulation of ANP synthesis and processing in atrial and extraatrial tissues.

Amino Acid Sequence↗

Microarray analysis of gene expression in human donor corneas.

OBJECTIVES: To use microarray analysis to identify genes expressed in human donor corneas and to create a preliminary, comprehensive database of human corneal gene expression. METHODS: A complementary DNA (cDNA) library was constructed from transplant-quality, human donor corneas. Biotin-labeled RNA was transcribed from the cDNA library and hybridized in duplicate to microarrays containing approximately 5600 human genes. Results were analyzed using a gene database of the National Institutes of Health, Bethesda, Md. Reverse transcriptase polymerase chain reaction analysis was performed to confirm corneal expression of genes identified by microarray analysis. RESULTS: Duplicate microarrays identified the expression of 1200 genes in human donor corneas. Chromosomal loci had been assigned to 1025 (85%) of these genes. A preliminary database of human corneal gene expression was compiled. A Web site containing these genes was created. Six collagen genes were identified that had not previously been localized within the cornea. Five apoptosis-related genes were identified, 4 of which had not previously been localized within the cornea. Three genes previously shown to cause corneal diseases were identified. Reverse transcriptase polymerase chain reaction analysis of genes identified by microarray analysis confirmed the corneal expression of 2 apoptosis-related genes and 1 collagen gene. CONCLUSIONS: Microarray analysis of healthy human donor corneas has produced a preliminary, comprehensive database of corneal gene expression. Large-scale analysis of gene expression has the potential to generate large amounts of data, which should be made readily accessible to the scientific community. The Internet offers many potential advantages as a medium for the maintenance of these large data sets. CLINICAL RELEVANCE: Identification of structural, apoptosis-related, and disease-causing genes within the cornea by microarrays may increase the understanding of normal and abnormal corneal function with likely relevance to corneal diseases and transplants.

Chromosomes, Human↗