Isolation and characterization of full-length functional cDNA clones for human carcinoembryonic antigen

Carcinoembryonic antigen (CEA) expression is perhaps the most prevalent of phenotypic changes observed in human cancer cells. The molecular genetic basis of this phenomenon, however, is completely unknown. Twenty-seven CEA cDNA clones were isolated from a human colon adenocarcinoma cell line. Most of these clones are full length and consist of a number (usually three) of surprisingly similar long (534 base pairs) repeats between a 5' end of 520 base pairs and a 3' end with three different termination points. The predicted translation product of these clones consists of a processed signal sequence of 34 amino acids, an amino-terminal sequence of 107 amino acids, which includes the known terminal amino acid sequence of CEA, three repeated domains of 178 amino acids each, and a membrane-anchoring domain of 27 amino acids, giving a total of 702 amino acids and a molecular weight of 72,813 for the mature protein. The repeated domains have conserved features, including the first 67 amino acids at their N termini and the presence of four cysteine residues. Comparisons with the amino acid sequences of other proteins reveals homology of the repeats with various members of the immunoglobulin supergene family, particularly the human T-cell receptor gamma chain. CEA cDNA clones in the SP-65 vector were shown to produce transcripts in vitro which could be translated in vitro to yield a protein of molecular weight 73,000 which in turn could be precipitated with CEA-specific antibodies. CEA cDNA clones were also inserted into an animal cell expression vector and introduced by transfection into mammalian cell lines. These transfectants produced a CEA-immunoprecipitable glycoprotein which could be visualized by immunofluorescence on the cell surface.

Download Full-text

Molecular cloning of the alpha-globin transcription factor CP2

Molecular and Cellular Biology ◽

10.1128/mcb.12.2.828-835.1992 ◽

1992 ◽

Vol 12 (2) ◽

pp. 828-835

Author(s):

L C Lim ◽

S L Swendeman ◽

M Sheffery

Keyword(s):

Amino Acids ◽

Molecular Weight ◽

Transcription Factor ◽

Amino Acid ◽

Core Region ◽

Cdna Clones ◽

Amino Terminal ◽

Human Cdna ◽

Alpha Globin ◽

P Domain

CP2, a transcription factor that binds the murine alpha-globin promoter, was purified and subjected to amino acid sequence analysis. Oligonucleotide primers derived from the sequence were used to obtain murine and human cDNA clones for the factor. The murine cDNA spans approximately 4 kb and contains two coextensive open reading frames (ORFs) which encode deduced polypeptides of 529 (ORF-1; molecular weight, 59,802) and 502 (ORF-2; molecular weight, 56,957) amino acids, slightly smaller than the purified factor as estimated from its mobility in sodium dodecyl sulfate-polyacrylamide gels (64,000 to 66,000). The human cDNA contains a single ORF of 501 amino acids that is nearly contiguous with murine ORF-2. Indeed, comparison of deduced human and murine amino acid sequences shows that the two polypeptides are 96.4% identical. A strictly conserved region is rich in serine and threonine (17.5%) and in proline (11%) residues (S-T-P domain). This S-T-P domain is immediately amino terminal to a string of 10 glutamines (in the human sequence) or a tract of alternating glutamine and proline residues (in the mouse sequence). Bacterial expression of the full-length (502-amino-acid) murine factor or of a core region comprising amino acids 133 to 395 generated polypeptides with the DNA binding specificity of CP2. These results confirmed the cloning of CP2 and delimited the region sufficient for specific DNA sequence recognition. Antisera produced against the core region recognized polypeptide species with Mrs of 64,000 and 66,000 in immune blots of nuclear extracts prepared from both murine and human cell lines, consistent with the size of the purified factor. Lastly, a data base search revealed that amino acids 63 to 270 of the murine factor are distantly related to a domain in the Drosophila gene regulatory factor Elf-1.

Download Full-text

Molecular cloning of the alpha-globin transcription factor CP2.

Molecular and Cellular Biology ◽

10.1128/mcb.12.2.828 ◽

1992 ◽

Vol 12 (2) ◽

pp. 828-835 ◽

Cited By ~ 59

Author(s):

L C Lim ◽

S L Swendeman ◽

M Sheffery

Keyword(s):

Amino Acids ◽

Molecular Weight ◽

Transcription Factor ◽

Amino Acid ◽

Core Region ◽

Cdna Clones ◽

Amino Terminal ◽

Human Cdna ◽

Alpha Globin ◽

P Domain

CP2, a transcription factor that binds the murine alpha-globin promoter, was purified and subjected to amino acid sequence analysis. Oligonucleotide primers derived from the sequence were used to obtain murine and human cDNA clones for the factor. The murine cDNA spans approximately 4 kb and contains two coextensive open reading frames (ORFs) which encode deduced polypeptides of 529 (ORF-1; molecular weight, 59,802) and 502 (ORF-2; molecular weight, 56,957) amino acids, slightly smaller than the purified factor as estimated from its mobility in sodium dodecyl sulfate-polyacrylamide gels (64,000 to 66,000). The human cDNA contains a single ORF of 501 amino acids that is nearly contiguous with murine ORF-2. Indeed, comparison of deduced human and murine amino acid sequences shows that the two polypeptides are 96.4% identical. A strictly conserved region is rich in serine and threonine (17.5%) and in proline (11%) residues (S-T-P domain). This S-T-P domain is immediately amino terminal to a string of 10 glutamines (in the human sequence) or a tract of alternating glutamine and proline residues (in the mouse sequence). Bacterial expression of the full-length (502-amino-acid) murine factor or of a core region comprising amino acids 133 to 395 generated polypeptides with the DNA binding specificity of CP2. These results confirmed the cloning of CP2 and delimited the region sufficient for specific DNA sequence recognition. Antisera produced against the core region recognized polypeptide species with Mrs of 64,000 and 66,000 in immune blots of nuclear extracts prepared from both murine and human cell lines, consistent with the size of the purified factor. Lastly, a data base search revealed that amino acids 63 to 270 of the murine factor are distantly related to a domain in the Drosophila gene regulatory factor Elf-1.

Download Full-text

Isolation and characterization of some novel genes of the apolipoprotein A-I family in Japanese eel, Anguilla japonica

Open Life Sciences ◽

10.2478/s11535-011-0042-8 ◽

2011 ◽

Vol 6 (4) ◽

pp. 545-557 ◽

Cited By ~ 2

Author(s):

Malay Choudhury ◽

Takahiro Oku ◽

Shoji Yamada ◽

Masaharu Komatsu ◽

Keita Kudoh ◽

...

Keyword(s):

Amino Acids ◽

Molecular Weight ◽

Amino Acid ◽

Consensus Sequence ◽

Structural Features ◽

Lipid Binding ◽

Japanese Eel ◽

Amino Acid Sequences ◽

Novel Genes ◽

Isolation And Characterization

AbstractApolipoproteins such as apolipoprotein (apo) A-I, apoA-IV, and apoE are lipid binding proteins synthesized mainly in the liver and the intestine and play an important role in the transfer of exogenous or endogenous lipids through the circulatory system. To investigate the mechanism of lipid transport in fish, we have isolated some novel genes of the apoA-I family, apoIA-I (apoA-I isoform) 1–11, from Japanese eel by PCR amplification. Some of the isolated genes of apoIA-I corresponded to 28kDa-1 cDNAs which had already been deposited into the database and encoded an apolipoprotein with molecular weight of 28 kDa in the LDL, whereas others seemed to be novel genes. The structural organization of all apoIA-Is consisted of four exons separated by three introns. ApoIA-I10 had a total length of 3232 bp, whereas other genes except for apoIA-I9 ranged from 1280 to 1441 bp. The sequences of apoIA-Is at the exon-intron junctions were mostly consistent with the consensus sequence (GT/AG) at exon-intron boundaries, whereas the sequences of 3′ splice acceptor in intron 1 of apoIA-I1-7 were (AC) but not (AG). The deduced amino acid sequences of all apoIA-Is contained a putative signal peptide and a propeptide of 17 and 5 amino acid residues, respectively. The mature proteins of apoIA-I1-3, 7, and 8 consisted of 237 amino acids, whereas those of apoIA-I4-6 consisted of 239 amino acids. The mature apoIA-I10 sequence showed 65% identity to amino acid sequence of apoIA-I11 which was associated with an apolipoprotein with molecular weight of 23 kDa in the VLDL. All these mature apoIA-I sequences satisfied the common structural features depicted for the exchangeable apolipoproteins such as apoA-I, apoA-IV, and apoE but apoIA-I11 lacked internal repeats 7, 8, and 9 when compared with other members of apoA-I family. Phylogenetic analysis showed that these novel apoIA-Is isolated from Japanese eel were much closer to apoA-I than apoA-IV and apoE, suggesting new members of the apoA-I family.

Download Full-text

A Novel Human Actin-Binding Protein Homologue That Binds to Platelet Glycoprotein Ib

Blood ◽

10.1182/blood.v92.4.1268.416k12_1268_1276 ◽

1998 ◽

Vol 92 (4) ◽

pp. 1268-1276 ◽

Cited By ~ 1

Author(s):

Wen-feng Xu ◽

Zhi-wei Xie ◽

Dominic W. Chung ◽

Earl W. Davie

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Binding Protein ◽

Full Length ◽

Actin Binding ◽

Intracellular Domain ◽

Platelet Surface ◽

Full Length Cdna ◽

Actin Binding Protein ◽

Amino Terminal

Glycoprotein (GP)Ib-IX-V is one of the major transmembrane complexes present on the platelet surface. Its extracellular domain binds von Willebrand factor (vWF) and thrombin, while its intracellular domain associates tightly with the cytoskeleton through the actin-binding protein (ABP)-280, also known as filamin. In the present study, a full-length cDNA coding for a human ABP homologue has been cloned and sequenced. This protein was identified by the yeast two-hybrid screening procedure via its interaction with the intracellular domain of GPIb. Initially, a 1.3-kb partial cDNA was isolated from a megakaryocyte-like cell line (K562) cDNA library followed by a full-length cDNA of 9.4 kb that was identified in a human placenta library. The full-length cDNA encoded a protein of 2,578 amino acids with a calculated molecular weight of 276 kD (ABP-276). The amino terminal 248 amino acids contained an apparent actin binding domain followed by 24 tandem repeats each containing about 96 amino acids. The amino acid sequence of the protein shared a high degree of homology with human endothelial ABP-280 (70% identity) and chicken filamin (83% identity). However, the 32 amino acid Hinge I region in ABP-280 that contains a calpain cleavage site conferring flexibility on the molecule, was absent in the homologue. An isoform containing a 24 amino acid insertion with a unique sequence at the missing Hinge I region was also identified (ABP-278). This isoform resulted from alternative RNA splicing. ABP-276 and/or ABP-278 were present in all tissues examined, but the relative amount varied in that some tissue contained both forms, while other tissue contained predominately one or the other. © 1998 by The American Society of Hematology.

Download Full-text

Trans-tegumental absorption of L-alanine and L-leucine by a monogenean, Diclidophora merlangi

Parasitology ◽

10.1017/s0031182000047363 ◽

1978 ◽

Vol 76 (1) ◽

pp. 29-37 ◽

Cited By ~ 21

Author(s):

D. W. Halton

Keyword(s):

Amino Acids ◽

Molecular Weight ◽

Amino Acid ◽

Sea Water ◽

Low Molecular Weight ◽

Acid Uptake ◽

Neutral Amino Acids ◽

Freeze Dried ◽

Significant Difference

SummaryAn in vitro investigation has been made of the relative roles of the gut and tegument in the absorption of the neutral amino acids L-alanine and L-leucine by a marine fish-gill parasite, Diclidophora merlangi. The use of ligatures to preclude oral ingestion of trace-labelled medium has proved inadequate, invariably damaging the tegument, as revealed by stereoscan electron microscopy, and resulting in artifactual levels of absorption. Three alternative procedures have given consistently reliable data on the route of entry of low molecular weight substrates. (1) Ultrastructural examination of worms previously incubated in electron-dense cationic tracers has shown that, in vitro, there is no oral intake of sea water. (2) The suspending of worms in trace-labelled medium with the mouth out of the medium and comparing amino acid uptake with that of worms totally immersed in medium has revealed no statistically significant difference in the absorption levels. (3) Application of section (freeze-dried) auto-radiography to detect diffusible isotope has demonstrated directly transtegumental absorption of a neutral amino acid. It is concluded from these experiments that Diclidophora has a tegumental transport system for absorbing certain neutral amino acids, and whilst, clearly, the worm is sanguinivorous and digests blood in a well-developed gut, it may also be capable of supplementing this diet with low molecular weight organic nutrient absorbed directly from sea water via the tegument.

Download Full-text

Nomenclature of Secreted Platelet Proteins – Report of the Working Party on Secreted Platelet Proteins of the Subcommittee on Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661294 ◽

1985 ◽

Vol 53 (02) ◽

pp. 282-284 ◽

Cited By ~ 10

Author(s):

Karen L Kaplan ◽

Stefan Niewiarowski

Keyword(s):

Amino Acids ◽

Molecular Weight ◽

Amino Acid ◽

Cellulose Acetate ◽

Working Party ◽

Platelet Factor 4 ◽

Cellulose Acetate Electrophoresis ◽

Platelet Factor ◽

Amino Terminal ◽

Platelet Proteins

SummaryStandard nomenclature for a number of secreted platelet proteins was agreed upon by The Working Party on Secreted Platelet Proteins of the Subcommittee on Platelets. Platelet factor 4 will continue to be used for the molecule with high heparin affinity, subunit molecular weight of 7780, and the described amino acid sequence. β-Thromboglobulin will be used to designate β-Thromboglobulin (81 amino acids/subunit, β-mobility on cellulose-acetate electrophoresis, pI 7), low-affinity platelet factor 4 (85 amino acids/subunit, y-mobility on cellulose-acetate electrophoresis, pI 8), and platelet basic protein (94 amino acids/ subunit, pI 10) when these are measured immunologically in plasma, but that thromboglobulin with a superscript designation of the pI should be used when assays are conducted on samples after isoelectric focusing, and a subscript amino-terminal amino acid can be added when a purified protein is described. Thrombospondin will continue to be the designation for the high molecular weight trimer that has previously been called thrombospondin or glycoprotein G. Platelet derived growth factor will be used for the group of closely related proteins of molecular weight about 30,000 and isoelectric point about 10.

Download Full-text

ISOLATION AND CHARACTERIZATION OF cDNA CLONES FOR THE PLATELET MEMBRANE GLYCOPROTEINS IIb and IIIa

10.1055/s-0038-1643961 ◽

1987 ◽

Author(s):

A E Butler-Zimrin ◽

J S Bennett ◽

M Poncz ◽

E Schwartz ◽

S Surrey ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Transmembrane Domain ◽

Platelet Membrane ◽

Membrane Receptors ◽

Cdna Clones ◽

Single Chain ◽

Platelet Protein ◽

Isolation And Characterization ◽

Terminal Amino

The platelet membrane GPIIb/GPIIIa complex on activated platelets contains receptors for fibrinogen, von Willebrand factor, and fibronectin. GPIIb and GPIlia also appear to be members of a family of membrane receptors involved in cell-cell and cell-matrix interactions. To study the structure of GPIIb and GPIIIa, we have constructed an expression library in the vector lambda gtll using mRNA from the HEL cell line and screened it with polyclonal antibody against each platelet protein. HEL cells constitutively express proteins similar to platelet GPIIb and GPIIIa. A 3.2kb GPIIb cDNA clone was identified that encodes for all 1008 amino acids of GPIIb including the known N-terminal amino acids of the α Cand βsubunits. This confirms that GPIIb is synthesized as a single chain polypeptide that is cleaved into two disulfide-linked subunits posttranslation. Analysis of the amino acid sequence revealed a major C-terminal transmembrane domain in the βsubunit, two potential transmembrane domains near the N-terminus of the αsubunit, and four possible N-linked glycosylation sites. Approximately 30% amino acid identity was found between GPIIb and the available amino acid sequences for the larger chains of the fibronectin and vitronectin receptors. Initial sequence analysis of a 3.8kb cDNA for GPIIIa included the known N-terminal amino acids of the platelet protein. Northern blot analysis was performed using HEL cell total RNA. The GPIIb cDNA hybridized to a 4.1kb mRNA while the GPIIIa cDNA hybridized to a 5.8kb mRNA. This indicates that the two cDNAs do not cross-hybridize and suggests that GPIIb and GPIIIa are encoded by separate genes. The availability of these cDNA for GPIIb and GPIIIa will facilitate study of the structure and function of the proteins and will aid in clarifying their relationship to other adhesive protein receptors.

Download Full-text

PAX8, a human paired box gene: isolation and expression in developing thyroid, kidney and Wilms’ tumors

Development ◽

10.1242/dev.116.3.611 ◽

1992 ◽

Vol 116 (3) ◽

pp. 611-623 ◽

Cited By ~ 8

Author(s):

A. Poleev ◽

H. Fickenscher ◽

S. Mundlos ◽

A. Winterpacht ◽

B. Zabel ◽

...

Keyword(s):

Amino Acid ◽

Expression Pattern ◽

Gene Isolation ◽

Human Thyroid ◽

Cdna Clones ◽

Rich Region ◽

Amino Terminal ◽

Isolation And Characterization ◽

Restricted Expression Pattern ◽

Pax8 Gene

Recent evidence indicates a crucial role for paired box genes in mouse and human embryogenesis. The murine Pax8 gene encodes a sequence-specific transcription factor and is expressed in the developing secretory system as well as in the developing and adult thyroid. This restricted expression pattern suggested involvement of the Pax8 gene in the morphogenesis of the above organs and prompted us to investigate the PAX8 gene in humans. In this report, we describe the isolation and characterization of PAX8 cDNAs from a human adult kidney cDNA library. An open reading frame of 450 amino acids contains the 128 amino acid paired domain at its amino-terminal end. The predicted human and mouse Pax8 proteins show 97.8% conservation and are identical in their paired domains. Two independent cDNA clones reveal differential splicing of the PAX8 transcripts resulting in the removal of a 63 amino acid serine-rich region from the carboxy end of the predicted Pax8 protein. The truncated Pax8 protein becomes more similar to the predicted murine Pax2 protein, that is also expressed during kidney development and lacks the serine rich region. RNAse protection analysis shows the presence of both PAX8 transcripts in human thyroid, kidney and five Wilms’ tumors. No truncated Pax8 transcripts could be detected in mouse kidney. In situ hybridization to sections of human embryonic and fetal kidney showed expression of PAX8 in condensed mesenchyme, comma-shaped and S-shaped bodies. In contrast, PAX2 expression was present mainly in the very early stages of differentiation, in the induced, condensing mesenchyme. This restricted expression pattern suggests a specific role for both genes during glomeruli maturation.(ABSTRACT TRUNCATED AT 250 WORDS)

Download Full-text

A mutant E2F-1 transcription factor that affects the phenotype of NIH3T3 fibroblasts inefficiently associates with cyclin A - cdk2

Biochemistry and Cell Biology ◽

10.1139/o98-015 ◽

1998 ◽

Vol 76 (1) ◽

pp. 37-44 ◽

Cited By ~ 5

Author(s):

Kelly L Jordan-Sciutto ◽

David J Hall

Keyword(s):

Cell Cycle ◽

Amino Acids ◽

Transcription Factor ◽

Cyclin A ◽

Chemotherapeutic Agents ◽

S Phase ◽

Full Length ◽

Vitro Assay ◽

Amino Terminal

The amino-terminal domain of the E2F1 transcription factor is the site of association with cyclin A - cdk2, mapping to residues 87-94. A mutant of E2F1 lacking the first 87 amino acids (termed E2F1d87) has a number of potent effects on cellular phenotype when constitutively expressed in NIH3T3 fibroblasts. For example, in these fibroblasts the duration of S phase and the sensitivity to S phase chemotherapeutic agents are both increased. Since E2F1d87 only partially truncates the cyclin A - cdk2 binding domain, it was important to determine the level of cyclin A - cdk2 association with this mutant to correlate any reduction in association with the observed effects on the cell cycle. It was found that cyclin A - cdk2 binds E2F1d87 in an in vitro assay but that this binding is reduced approximately 8 fold compared with binding to full-length E2F1, whereas no detectable binding was seen to a mutant E2F1 that lacks the first 117 amino acids. Correspondingly, H1 kinase activity in E2F1d87 immunoprecipitates from E2F1d87-expressing cells was significantly reduced compared with that seen for full-length E2F1. From these data it appears that E2F1 with reduced cyclin A - cdk2 binding activity mediates the alteration in cell cycle parameters seen in these cells.Key words: E2F1, apoptosis, cyclin A, cell cycle.

Download Full-text