Pregnancy-specific beta 1 glycoprotein in rat: tissue distribution of the mRNA and identification of testicular cDNA clones

A partial cDNA of pregnancy-specific beta 1 glycoprotein isolated from human term placenta was used as probe for slot-blot analysis of total RNA extracted from placental and non-placental tissues in the rat. RNA hybridization with the probe was observed in rat placenta, indicating the presence of mRNA highly homologous to human SP1. The quantity of hybridizing RNA increased with increasing gestational age. In non-pregnant rats, SP1-hybridizing mRNAs were found in uterus, intestine and testis, while no hybridizing material was detected in liver or muscle. The amount of rat SP1 mRNA, based on percentage of total tissue RNA, was greatest in the testis followed by intestine, uterus and placenta. Using the same probe, six clones were obtained by screening a rat testis cDNA library. These clones carried cDNA inserts ranging in size from 1530 to 1983 bp. An internal EcoRI site was present in all cDNA clones. Southern blot analysis confirmed that the cDNA insert of all the clones was homologous to human placental SP1 cDNA. These results suggest a possible origin for the trace quantities of SP1 detected in non-pregnant individuals. It also confirms that the rat is an appropriate model for studying the physiological functions of SP1.     

Determination of the molecular nature and cellular localization of Thy-1 in human renal tissue

The molecular nature and cellular localization of Thy-1 antigen in human renal tissue were studied. Strong immunohistochemical staining was observed in frozen sections of human kidney using monoclonal anti-human Thy-1 antibody; this reaction was almost completely abolished by pretreating the kidney section with phosphatidyl inositol (PI)-specific phospholipase C (PI-PLC). Immunohistochemical analysis revealed that the Thy-1 antigen is localized on the proximal tubular epithelial cells and the Bowman's capsule of the glomerulus. Northern blot analysis of renal mRNA using a cloned human Thy-1 gene revealed the presence of human Thy-1 mRNA of a similar size to the one in human brain. When a human kidney cDNA library was screened with the same probe, a cDNA of human Thy-1 was isolated. Moreover, human Thy-1 protein with a molecular weight (MW) of 21,000 was detected in renal tissue by gel electrophoresis and Western blot analysis using monoclonal anti-human Thy-1 antibody. These data demonstrate for the first time the production of human Thy-1 as a PI-anchored protein with a unique cellular location in human renal tissue.     

Structure and expression of osteonectin mRNA in human tissue

A cDNA encoding osteonectin was isolated from a human bone cell cDNA library and used to examine osteonectin protein structure, mRNA structure and expression in human tissue. The deduced protein sequence shows complete identity with a recently isolated placental form and extensive homology to mouse and bovine counterparts. The protein is rich in cysteine residues, which are conserved between species except for cys 194 which is only present in the bovine. In the human, osteonectin mRNA is of two sizes, 2.3 and 3.0 kb, the former being dominant in all tissues studied. Human mRNA was detected in the Ewing sarcoma and in non-bone cell and tissue sources. The potential folded structure of osteonectin mRNA was estimated, based on computer predictions, and indicates the presence of a bulge at the 5' end of the message which includes the start of translation. Southern analysis of human genomic DNA using radiolabeled osteonectin cDNA as probe demonstrates a simple banding pattern confirming earlier studies that the osteonectin gene is present in one copy per haploid human genome.     

差异筛选肝癌凋亡细胞cDNA文库的简便方法

目的差异筛选人肝癌凋亡细胞ｃＤＮＡ文库。方法消减杂交和点杂交相结合的噬菌斑原位杂交法筛选ｃＤＮＡ文库，首先采用（－）ｃＤＮＡ探针杂交，挑取（－）的噬菌斑克隆，再用（－）和（＋）两种ｃＤＮＡ探针与初筛出的噬菌斑克隆杂交的差异筛选方法。结果得到４个充分孤立的噬菌斑克隆，其插入片段长度为１．５ｋｂ左右。结论该方法简便、快速，是差异筛选ｃＤＮＡ文库的一种较为可行的简便方法。     

Cloning and expression of a chitinase gene from the hyperparasitic fungus Aphanocladium album

Summary Recombinant clones from a cDNA library of an Aphanocladium album chitinase-overproducing mutant strain were isolated by screening with antiserum against a 39 kDa chitinase purified from this hyperparasitic fungus. Analysis of the isolated positive clones indicated that most of them carried the same cDNA. A cDNA from this group was used as a hybridization probe to isolate an 8 kb DNA fragment from a genomic library of the wild-type strain. The chitinase 1 gene was mapped to this fragment by two independent approaches. Its partial DNA sequence was in perfect agreement with an amino-terminal peptide sequence obtained by sequencing 23 amino acids of the 39 kDa chitinase. Its transfer in Fusarium oxysporum resulted in a transformant producting both a protein of about 39 kDa that cross-reacted with the chitinase antiserum and a chitinase activity that was inhibited by the same antiserum. Northern blot analysis indicates that the cloned chitinase gene was subject to catabolite repression and appeared inducible by chitin.     

Comparison of the canine and human acid β-galactosidase gene

Several canine cDNA libraries were screened with human β-galactosidase cDNA as probe. Seven positive clones were isolated and sequenced yielding a partial (2060 bp) canine β-galactosidase cDNA with 86% identity to the human β-galactosidase cDNA. Preliminary analysis of a canine genomic library indicated conservation of exon number and size. Analysis by Northern blotting disclosed a single mRNA of 2.4 kb in fibroblasts and liver from normal dogs and dogs affected with GM1 gangliosidosis. Although incomplete, these results indicate canine GM1 gangliosidosis is a suitable animal model of the human disease and should further efforts to devise a gene therapy strategy for its treatment. © 1996 Wiley-Liss, Inc.     

A portion of the human surfactant protein A (SP-A) gene locus consists of a pseudogene

SP-A is the most abundant, surfactant-associated protein isolated from lung lavage. Genomic blot analysis of total human cellular DNA with SP-A cDNA demonstrated the presence of multiple hybridizing fragments that are not accounted for by available SP-A gene sequences. In this report, we have cloned and characterized human genomic DNA fragments that account for some of the other hybridizing fragments. These clones contain nucleotide sequences that are highly homologous to the fourth intron and fifth exon of the human SP-A gene. Sequences upstream from these SP-A-like sequences are not detectable by Northern blot hybridization of SP-A-expressing cells and the SP-A-like sequences contain premature stop codons, consistent with the interpretation that these clones represent an SP-A pseudogene. Restriction fragments consistent with this pseudogene and the functional SP-A gene are present in a human chromosome 10 genomic library made from a single chromosome, showing that the functional SP-A gene and the pseudogene are syntenic.     

Molecular cloning, expression and characterization of the ovine IL-2R alpha chain.

Interleukin-2 (IL-2) stimulates the proliferation of activated antigen-specific T cells through its interaction with high affinity receptors. This event is largely regulated by the inducible expression of the alpha-chain (CD25) which, in combination with the beta-chain and possibly additional chains, forms the high affinity IL-2 receptor (IL-2R) complex. From a concanavalin A (Con A)-activated ovine T-cell complementary DNA (cDNA) library we have isolated two cDNA clones which together constitute a 2650 base pair (bp) messenger RNA (mRNA) species encoding the ovine IL-2R alpha chain. The nucleotide sequence has high homology with analogous cDNA from other species and predicts a mature protein of 254 amino acids. In addition to the predominate 2.6 kilobase (kb) ovine IL-2R alpha chain mRNA species. Northern blot analysis of activated T-cell RNA revealed two larger mRNA species. The ovine IL-2R alpha chain cDNA was transfected into CHO cells and low affinity binding of human recombinant IL-2 demonstrated. Polyclonal antisera generated against the transfected cells cross-reacted with Con A-activated ovine lymphocytes. In addition these antisera were used to immunoprecipitate a unique 50,000 MW protein from the transfected cells. It is likely that this protein represents the expressed ovine IL-2R alpha chain cDNA which is heavily glycosylated as distinct from the 30,869 MW primary translation product. Southern blot analysis of ovine genomic DNA suggests that the ovine IL-2R alpha chain is encoded by a single copy gene.     

Isolation of cDNA clones and genomic DNA clones of beta-subunit of chicken prolyl 4-hydroxylase

Prolyl 4-hydroxylase (EC 1.14.11.2) is a key enzyme in collagen biosynthesis. The active enzyme is a tetramer composed of two pairs of non-identical subunits, alpha and beta. Sheep antiserum directed against chicken proly 4-hydroxylase was initially used to screen two cDNA expression libraries. The cDNA was prepared from chicken smooth muscle mRNA and cloned into the plasmids pUC8- and pUC9. Antibodies identified twenty-five clones among the approximately 2 x 10(5) clones in the libraries. Ten clones were isolated pure and used in the subsequent analysis. Monospecific antibodies directed against beta subunit of the enzyme were used in Western-blot analyses of extracts of bacteria carrying the cDNA clones. The results indicated that the clone CPH 9-10B encodes a portion of beta-subunit. The cDNA from CPH 9-10B was used to screen another cDNA library prepared from mRNA from chicken skeletal muscle. Several overlapping clones were isolated. Together the cDNAs correspond to 2.4 kb which is the same as the corresponding mRNA. Three regions of the amino acid sequence deduced from the cDNA sequence matched with that of the NH2-terminus of beta-subunit and two CNBr peptides derived from beta-subunit. The cDNA of CPH 9-10B was also used to screen a genomic DNA library constructed with lambda EMBL-3. Two overlapping genomic clones lambda gCPH beta-22 and beta-50 were isolated and characterized by restriction enzyme analysis. The results indicate that lambda gCPH beta-22 contains the portion of the beta-subunit gene that is transcribed into the 5' portion of beta-subunit mRNA, whereas lambda gCPH beta-50 contains the 3' portion.     

Molecular characterisation of a putative endogenous retrovirus cDNA isolated from human placental tissue

The human genome comprises of abundant DNA sequences related to endogenous retroviruses (ERV) and a variety of solitary long terminal repeats (LTRs). Substantial numbers of intact retroviral particles have been detected by electron microscopy in normal human placental villous tissue particularly in syncytiotrophoblast. Understanding the molecular structure, organisation and distribution of these ERV sequences may lead to elucidation of their possible dual function at the foetal-maternal interface; proliferation and differentiation of cytotrophoblast and induction of local pregnancy-associated immune suppression thus allowing survival of the foetal allograft. In this study, antibody probes were used to screen a human placental expression library and cDNA clones isolated were characterized by polymerase chain reaction, Southern blot hybridisation, DNA cloning and partial nucleotide sequencing. A specific 1.7kb-cDNA clone was isolated from a human placental expression library. Further characterisation showed this clone represents a single copy gene, approximately 9-10kb and did not hybridise to the env region of ERV3 human endogenous retrovirus. The 1.7kb-cDNA clone may represent a provirus co-expressed with cellular sequences.     

Characterization of a Tryptase mRNA Expressed in the Human Basophil Cell Line KU812

The expression of a tryptic serine protease was detected in the cell line KU812 by Northern blot analysis with an oligonucleotide probe directed against a conserved region present in all of the five presently cloned human mast cell tryptases. PCR primers designed for the amplification of a nearly full-length copy of tryptase mRNAs were used to study the identity of the KU812 tryptase. Ten clones were characterized and all were found to be identical to one of the tryptases previously cloned from a human skin cDNA library. This tryptase has been thought to originate from mast cells of the skin.
Two possible explanations may account for the observed identity between the presumed mast cell tryptase and the KU812 tryptase. Firstly, it is possible that the KU812 tryptase is a basophil-specific tryptase which has previously been cloned from a human skin cDNA library containing low levels of cDNA copies derived from basophils in the starting material. Secondly, the KU812 cell line, and possibly normal basophils, express a tryptase which is identical to one of the tryptases expressed in normal skin mast cells. We cannot at present rule out any of the two possibilities, but we favour the second explanation as being the most likely.     

Cloning and identification of genes differentially expressed in metastatic and non-metastatic rat rhabdomyosarcoma cell lines

The objective of this study was to identify genes involved in invasion and metastasis using a rat rhabdomyosarcoma model (SMF-A and RMS-B cell lines). The SMF-A cell line was established from a metastatic nodule of an induced rhabdomyosarcoma in syngeneic F344 rats. Two cell lines with defined metastatic potentials, SMF-Ai and SMF-Da, were cloned from the SMF-A line. The cell line SMF-Ai is tumorigenic, highly invasive and highly metastatic. On the other hand, the revertant line SMF-Da is less tumorigenic, non-invasive and non-metastatic. We have isolated from a SMF-Ai cDNA library eight cDNA clones which are differentially expressed by the metastatic SMF-Ai and the non-metastatic SMF-Da cell line using Northern blot analysis. Five of these clones, smf-4, smf-6, smf-41, smf-42 and smf-44, are overexpressed in the SMF-Da cell line and have homology with beta-2-microglobulin, lactate dehydrogenase, ribosomal protein L38, ribosomal protein S4 and acidic ribosomal phosphoprotein P1, respectively. The three other clones, smf-7, smf-40 and smf-61, are overexpressed in SMF-Ai. Clones smf-40 and smf-61 show significant homology with the human TB3-1 gene and the human fus gene respectively. The clone smf-7 has no significant homology with known sequences. We also analyzed the expression of these clones in other rat rhabdomyosarcoma cell lines (RMS-B and their clones) and in tumors obtained by injection of these cell lines into rats or nude mice. Smf-61 and smf-7 were the only clones with a differential expression pattern associated with the invasive or metastatic potential of all cell lines examined. A preliminary study of the expression of smf-7 and smf-61 in other cancer cell lines also showed mRNA expression in two human rhabdomyosarcomas and a human epidermoid carcinoma suggesting the existence of genes homologous to smf-7 and smf-61 clones in human cancers. Our findings suggest an association between the expression of smf-7 and smf-61 and invasive or metastatic potential of rhabdomyosarcoma cells.     

Isolation of genomic clones homologous to transcribed sequences from human X chromosome

Several X chromosome DNA clones homologous to transcribed sequences were isolated from a human X chromosome library. The clones were selected for their ability to hybridize either with³²P -labeled human cDNA in the presence of an excess of unlabeled human repetitive DNA or with mouse fibroblast cDNA. The X chromosome specificity of these sequences was demonstrated by two criteria: A dosage effect was seen when the clones were hybridized to Southern blots of DNA from 1X and 5X cells, and they hybridized to DNA from mouse-human hybrid cells containing only the human X chromosome. The presence of transcribed sequences in these X clones was detected by hybridization with mouse cDNA or with human cDNA in the presence of unlabeled human repetitive sequences, by identifying restriction fragments which hybridize with cDNA but not with human repetitive DNA, and by hybridization with poly A⁺ RNA on Northern blots. These clones were mapped on the human X chromosomes using a panel of mouse-human somatic cell hybrids carrying various translocated human X chromosomes.