Cloning of cDNA for a novel fibrinogen/angiopoietin-related protein, FARP

Using a low abundant gene screening strategy in the human dermal papilla cell cDNA library, we isolated a novel cDNA, which was 1,872 bp of nucleotides in length and contained an open reading frame encoding 405 amino acids. We designated it 'fibrinogen/angiopoietin-related protein' (FARP) as it contained the characteristic coiled-coil domain and fibrinogen-like domain in the NH2- and COOH-terminal, which are conserved in angiopoietins. FARP has a highly hydrophobic region at the N-terminus that is typical of a secretory signal sequence. Recently, a very similar gene, HFARP, was cloned and they have a difference of only 18 amino acids in N-terminus. While HFARP was expressed only in the liver, northern blot analysis showed that FARP mRNA is abundantly expressed in the liver, placenta, prostate, and ovary in human adult tissues. It was also expressed in the fetal liver and lung carcinoma cell line. Further study will be needed to clarify the function of the FARP gene.     

Rat KC cDNA cloning and mRNA expression in lung macrophages and fibroblasts.

We have isolated and sequenced overlapping cDNA clones for rat KC*. The 0.93 kb cDNA has a single open reading frame of 288 nucleotides, and substantial sequence identity with the platelet-factor 4 family members mouse KC, hamster gro, and human gro. Using cloned cDNA as a probe, expression of KC mRNA in lavaged rat alveolar macrophages (AMs) increased after lipopolysaccharide (LPS) treatment. We also studied expression in vitro by a rat fetal lung fibroblast cell line, RFL-6. Expression of KC mRNA in RFL-6 cells increased after treatment with interleukin 1 or with conditioned medium from rat AMs treated with LPS.     

Cloning of the human cDNA for the U1 RNA-associated 70K protein.

Anti-RNP sera were used to isolate a cDNA clone for the largest polypeptide of the U1 snRNP, a protein of mol. wt 70 kd designated 70K, from a human liver cDNA library constructed in the expression vector pEX1. The cro-beta-galactosidase-70K fusion protein reacted with various anti-RNP patient sera, a rabbit anti-70K antiserum, as well as with a monoclonal antibody specific for this protein. The sequences of four 70K peptides were determined and they match parts of the deduced amino acid sequence of the 1.3 kb insert of p70.1 indicating that it is a genuine 70K cDNA. Screening of a new cDNA library constructed from polysomal mRNA of HeLa cells with the p70.1 clone yielded an overlapping clone, FL70K, which was 2.7 kb long and covered the complete coding and 3'-untranslated sequence of the 70K protein in addition to 680 nucleotides upstream of the putative initiation codon, The predicted mol. wt of the encoded protein is approximately 70 kd. Amino acid analysis of the purified HeLa 70K protein yielded values close or identical to those deduced from the nucleotide sequence of the full-length cDNA. The 70K protein is rich in arginine (20%) and acidic amino acids (18%). Extremely hydrophilic regions containing mixed-charge amino acid clusters have been identified at the carboxyl-terminal half of the protein, which may function in RNA binding. A sequence comparison with two recently cloned RNA binding proteins revealed homology with one region in the U1 RNP 70K protein. This domain may also be responsible for RNA binding.     

Androgen Receptor Accelerates Premature Senescence of Human Dermal Papilla Cells in Association with DNA Damage

The dermal papilla, located in the hair follicle, expresses androgen receptor and plays an important role in hair growth. Androgen/Androgen receptor actions have been implicated in the pathogenesis of androgenetic alopecia, but the exact mechanism is not well known. Recent studies suggest that balding dermal papilla cells exhibit premature senescence, upregulation of p16^INK4a, and nuclear expression of DNA damage markers. To investigate whether androgen/AR signaling influences the premature senescence of dermal papilla cells, we first compared frontal scalp dermal papilla cells of androgenetic alopecia patients with matched normal controls and observed that premature senescence is more prominent in the dermal papilla cells of androgenetic alopecia patients. Exposure of androgen induced premature senescence in dermal papilla cells from non-balding frontal and transitional zone of balding scalp follicles but not in beard follicles. Overexpression of the AR promoted androgen-induced premature senescence in association with p16^INK4a upregulation, whereas knockdown of the androgen receptor diminished the effects of androgen. An analysis of γ-H2AX expression in response to androgen/androgen receptor signaling suggested that DNA damage contributes to androgen/androgen receptor-accelerated premature senescence. These results define androgen/androgen receptor signaling as an accelerator of premature senescence in dermal papilla cells and suggest that the androgen/androgen receptor-mediated DNA damage-p16^INK4a axis is a potential therapeutic target in the treatment of androgenetic alopecia.     

Isolation and characterization of mouse MUC18 cDNA gene, and correlation of MUC18 expression in mouse melanoma cell lines with metastatic ability

The cell surface adhesion molecule human MUC18 (huMUC18 or Mel-CAM) has been postulated to play a key pathogenic role in metastatic melanoma progression. To establish an immunocompetent syngeneic mouse model that would greatly facilitate our understanding of the role of MUC18 in the metastatic behavior of melanoma, we cloned and characterized the mouse MUC18 (muMUC18) cDNA gene. The gene was amplified by RT-PCR and RACE of the poly(A)+RNA isolated from the mouse melanoma cell line B16F10/Queens. The cloned muMUC18 cDNA gene contained 28 nucleotides of 5'-UTR, 908 nucleotides of 3'-UTR, and an open reading frame (ORF) of 1947 nucleotides encoding a protein of 648 amino acids, which is two amino acids longer than huMUC18. The size of the muMUC18 mRNA is about 3 kb with a shorter 3'-UTR than the huMUC18 mRNA (about 3.3 kb). Besides, the sequence in the 3' UTR of the two mRNAs is diverse with only 31% identity. The 5'-UTR and coding sequences of the muMUC18 cDNA are 72.4 and 80.6% identical to those of huMUC18, respectively. The deduced amino acid sequence of the muMUC18 cDNA is 76.2% identical to that of huMUC18. The amino acid sequences deduced from MUC18 cDNA sequences from six other mouse melanoma cell lines are identical except one to three residues, suggesting that the muMUC18 cDNA sequence determined in this report is correct. The muMUC18 protein is predicted to be slightly more acidic than the human protein. The levels of muMUC18 mRNA and protein in nine mouse melanoma cell lines were directly proportional to their ability to establish metastatic colonies in lungs of syngeneic mice. Most biological functions of the muMUC18 may be similar to the huMUC18.     

Sequence and expression of 11β-hydroxysteroid dehydrogenase type 1 cDNA cloned from pig testis

Pig 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1 cDNA was cloned from neonatal pig testis, and 15 nucleotides were found to differ from the sequence in GenBank (Accession No. NM_214248). It was an exclusive clone obtained as pig 11β-HSD type 1, and the sequence of 11β-HSD type 1 cDNA cloned from pig liver was identical to that from testis. Amino acid sequence, deduced from cloned cDNA, also had a conserved triad of catalytically important Ser, Tyr and Lys residues for the short-chain dehydrogenase/reductase family, a membrane-spanning domain consisting of hydrophobic amino acid and a glycine motif in the cofactor binding region. The protein translated from this clone on expression in mammalian HEK293 cells exhibited oxo-reduction activity of cortisone and oxidation activity of cortisol. Furthermore, this oxo-reduction activity of cortisone was stimulated by co-expression of human H6PDH, while oxidation activity of cortisol was suppressed by H6PDH co-expression in HEK293 cells. Based on these results, the sequence of newly cloned cDNA is considered to correspond to an active enzyme form of pig 11β-HSD type 1.     

Molecular cloning, complementary deoxyribonucleic acid structure and predicted full-length amino acid sequence of the hormone-inducible regulatory subunit of 3'-5'-cyclic adenosine monophosphate-dependent protein kinase from human testis

In this study, we report the isolation and characterization of a full-length cDNA clone for the hormone-inducible regulatory subunit RII beta (formerly called RII51) of type II cAMP-dependent protein kinase from a human testis cDNA library. The cloned cDNA demonstrated tissue-specific expression of RII beta mRNA in human tissues, with the highest mRNA levels in testis and ovary. The isolated human cDNA clone was 3.3 kilobases (kb) in length and contained 166 base pairs (bp) of G/C-rich 5'-noncoding sequence, an open reading frame of 1254 bp and an A/T-rich 3'-nontranslated region containing 1836 bp followed by an 89 nucleotide long poly(A)-tail. The predicted protein contains 418 amino acids including the start methionine, and the estimated mol wt of human RII beta is 53,856. The nucleotide sequence within the open reading frame and the predicted amino acid sequence of human RII beta are highly conserved compared with partial rat RII beta sequences, displaying 91% and 97% similarity, respectively. Codon preference analysis of the cloned cDNA sequence indicated that the two cAMP-binding domains and the hinge region are highly conserved through evolution, whereas the dimerization domain displayed a codon preference pattern indicative of appearance at a later stage of evolution. The isolated human cDNA detected an FSH- and cAMP-inducible mRNA of 3.2 kb in rat Sertoli cells, thus confirming that the cloned cDNA represents the hormone-inducible regulatory subunit of cAMP-dependent protein kinase. This is the first report documenting the isolation of a full-length cDNA clone for the RII beta of cAMP-dependent protein kinase.     

IGF1和IGF1R在小鼠第一个毛囊生长周期皮肤的表达

毛囊生长周期中,真皮乳头和毛基质间的基质 上皮信号调控细胞的增殖和分化。多功能细胞调控因子胰岛素样生长因子1（IGF1)是该信号路径的成员之一。第1个毛囊生长周期决定着毛囊的正常生长和发育,但IGF1在此期的作用未见报道。实时荧光定量PCR结果显示,IGF1在生长期皮肤中的相对表达量最低,在退化期表达量最高,在静止期表达量又降低。与生长初期相比,IGF1在退化期和静止期的表达量呈差异极显著（P＜0.01）;胰岛素样生长因子1受体（IGF1R）在生长期皮肤中的相对表达量最高,在退化期表达量最低,而在静止期表达量又升高。与生长初期相比,IGF1R在退化期和静止期的表达量呈差异极显著（P＜0.01）。Western 印迹结果显示,IGF1和IGF1R蛋白在小鼠皮肤第1个毛囊生长周期各阶段的表达趋势分别与其mRNA的表达趋势一致;免疫组织化学结果表明,IGF1主要分布在小鼠表皮,而IGF1R免疫阳性在小鼠毛囊毛球部、内外根鞘和毛乳头均有分布。以上实验结果揭示,IGF1和IGF1R在小鼠皮肤第1个毛囊生长周期的各阶段的差异性表达,可能在毛囊生长周期各阶段的转化过程中参与了黑色素的形成。然而,IGF1和IGF1R表达趋势不一致,提示IGF1在小鼠皮肤中发挥作用时,并非只与IGF1R结合才能发挥作用。     

The hair follicle: a paradoxical androgen target organ

Androgens are the main regulator of normal human hair growth. After puberty, they promote transformation of vellus follicles, producing tiny, unpigmented hairs, to terminal ones, forming larger pigmented hairs, in many areas, e.g. the axilla. However, they have no apparent effect on the eyelashes, but can cause the opposite transformation on the scalp leading to the replacement of terminal hairs by vellus ones and the gradual onset of androgenetic alopecia. This paradox appears to be an unique hormonal effect. Hair follicles are mainly epithelial tissues, continuous with the epidermis, which project into the dermis. A mesenchyme-derived dermal papilla enclosed within the hair bulb at the base controls many aspects of follicle function. In the current hypothesis for androgen regulation, the dermal papilla is also considered the main site of androgen action with androgens from the blood binding to receptors in dermal papilla cells of androgen-sensitive follicles and causing an alteration of their production of paracrine factors for target cells e.g. keratinocytes. Studies of cultured dermal papilla cells from sites with different responses to androgens in vivo have confirmed the paradoxical responses. All dermal papilla cells from androgen-sensitive sites contain low capacity, high affinity androgen receptors. However, only some cells formed 5alpha-dihydrotestosterone, e.g. beard but not axillary cells, in line with hair growth in 5alpha-reductase deficiency. Incubation with androgens also stimulated the mitogenic capacity of beard cell media, but inhibited that produced by scalp cells. This suggests that the paradoxical differences are due to differential gene expression within hair follicles, presumably caused during embryogenesis.     

A retinoic acid-inducible mRNA from human erythroleukemia cells encodes a novel tissue transglutaminase homologue.

A 1.9-kilobase (kb) cDNA for a new transglutaminase protein has been cloned and sequenced from retinoic acid-induced human erythroleukemia (HEL) cells. Full-length cDNA analysis reveals an open reading frame coding for a polypeptide of 548 amino acid residues with a molecular weight of 61,740. The deduced amino acid sequence exhibited 98% identity to the human cellular transglutaminase sequence. The cysteine at position 277 in the active site and the putative Ca(2+)-binding pocket at residues 446-453 of cellular transglutaminase are conserved. Such evidence predicts that the encoded protein product is likely to be a transglutaminase homologue (TGase-H). Immunoprecipitation of the in vitro translation products from a synthetic TGase-H mRNA and from total protein of cultured erythroleukemia HEL cells revealed a protein with a molecular weight of 63,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Northern blot analysis of HEL cells and normal human fibroblast cells WI-38 using a cellular TGase probe detected the 1.9- and 4.0-kb RNA species at a relative abundance of 1:3 and 1:7, respectively. The 3'-end of the human cellular transglutaminase mRNA was also cloned and sequenced to allow comparison to the 3'-end of TGase-H reported here. This new piece gives a full length of 4012 nucleotides (4.0 kb) for human cellular transglutaminase. Comparison of the 5'-end (bases 1-1747) of the 1.9- and 4.0-kb cDNA sequences revealed a very high degree of identity. Beginning with base 1748, the sequences diverge showing no homology. The divergence point correlates with known intron-exon consensus boundaries indicative of alternative splicing.     

Molecular cloning and characterization of the complementary DNA and gene coding for the B-chain of subcomponent C1q of the human complement system.

Plasmid clones containing cDNA coding for the B-chain of human Clq were isolated from a liver cDNA library. The longest cDNA insert isolated contained all the coding sequence for amino acid residues B1 to B226 plus a 3' non-translated region of 264 nucleotides that extended into the poly(A) tail, thus accounting for 950 nucleotides of the mRNA. The B-chain mRNA was estimated by Northern-blot analysis to be 1.46 kb (kilobases) long, which indicated that approx. 500 bases were not accounted for in the cDNA clone. A cosmid clone containing the C1q-B chain gene was isolated from a human genomic DNA library. The precise 5' limit of gene was not established, but from the data available it appears that the gene is approx. 2.6 kb long. The coding sequence for residues B1 to B226 in the gene is interrupted by one intron, of 1.1 kb, which is located within the codon coding for glycine at position B36. This glycine residue is located in the middle of the triple-helical regions found in C1q at exactly the position where there is an unusual structural feature, i.e. a bend in each of the helical regions brought about by the interruption of the Gly-Xaa-Yaa repeating triplet sequences in the A- and C-chains and the presence of an 'extra' triplet in the B-chain. Nucleotide sequencing of the 5' end of the gene indicates the presence of a predominantly hydrophobic stretch of 29 amino acids, immediately before residue B1, which could serve as a signal peptide.     

The structure and expression of the murine wildtype p53-induced phosphatase 1 (Wip1) gene

The human wildtype p53-induced phosphatase 1 (Wip1; GenBank symbol Ppm1d) gene encodes a type 2C protein phosphatase (PP2C) that is induced by ionizing radiation in a p53-dependent manner. We have cloned and sequenced the mouse Wip1 gene and its encoded mRNA. The mouse Wip1 gene is composed of six exons and spans over 36 kb of DNA. The mouse cDNA sequence predicts a 598-amino-acid protein with a molecular mass of roughly 66 kDa. Comparison of human and mouse Wip1 sequences revealed 83% overall identity at the amino acid level. The 5'-flanking region of exon 1 had promoter elements characteristic of a housekeeping gene. The Wip1 coding sequences share conserved functional regions with other PP2Cs from a diverse array of species. Expression of Wip1 mRNA was detected ubiquitously in adult and embryonic tissues, though expression in the testis was much higher than in other tissues. Wip1 has been mapped near the p53 gene on mouse chromosome 11.     

Induction of transforming growth factor-beta 1 by androgen is mediated by reactive oxygen species in hair follicle dermal papilla cells

The progression of androgenetic alopecia is closely related to androgen-inducible transforming growth factor (TGF)-β1 secretion by hair follicle dermal papilla cells (DPCs) in bald scalp. Physiological levels of androgen exposure were reported to increase reactive oxygen species (ROS) generation. In this study, rat vibrissae dermal papilla cells (DP-6) transfected with androgen receptor showed increased ROS production following androgen treatment. We confirmed that TGF-β1 secretion is increased by androgen treatment in DP-6, whereas androgeninducible TGF-β1 was significantly suppressed by the ROSscavenger, N-acetyl cysteine. Therefore, we suggest that induction of TGF-β1 by androgen is mediated by ROS in hair follicle DPCs. [BMB Reports 2013; 46(9): 460-464]     

Genetic transfer of a functional human interferon alpha receptor into mouse cells: cloning and expression of its cDNA

A cDNA coding for the human interferon alpha receptor has been cloned using a gene transfer approach. This consists of transferring human DNA to mouse cells and selecting for cells sensitive to human interferon alpha. The transfected cells expressed the human interferon alpha receptor, and a 5 kb human DNA was isolated from a secondary transfectant. This DNA defects an mRNA present in human cells and was used to clone a 2.7 kb cDNA from a library constructed from human Daudi cells. The sequence of the cDNA is presented. It codes for a glycoprotein of 557 amino acids with an N-terminal hydrophobic region and a single transmembrane-spanning segment. Mouse cells expressing the cDNA become sensitive to the antiviral activity of and express binding sites for human interferon alpha, demonstrating that the cloned cDNA encodes a functional human interferon alpha receptor.