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PubMed1990

Three human homologs of a murine gene encoding an inhibitor of stem cell proliferation.

The G0S19 genes are members of the "small inducible" family of genes, which have similar exon-intron organizations and encode secreted proteins with similar dispositions of cysteine and proline residues. G0S19-1 mRNA is increased shortly after the addition of lectin or cycloheximide to cultured human blood mononuclear cells. The cDNA sequence is homologous to that of a murine gene encoding an inhibitory cytokine (MIP1 alpha/SCI), which decreases hemopoietic stem cell proliferation. The homology extends to the 3' noncoding region, which contains two conserved elements: (i) GGGACTCTTC, a potential transcription factor NF chi B-binding site, and (ii) TTTTGTAATTTATTTT, which is found in some related genes (e.g., that encoding the immediate early protein ornithine decarboxylase). A similar but complementary sequence is present in human immunodeficiency virus. Two of the three human genes that hybridize to G0S19-1 cDNA were sequenced. G0S19-1 has 5' AP1-like recognition elements as found in some other phorbol ester-responsive genes (e.g., c-fos). G0S19-2 has a 5' Alu sequence, but is likely to be expressed because of the conservation of sections of the gene believed to be important for function. The 5' flanks of both genes contain the nucleotide motifs CK-2 and SRE, indicating cytokine-like genes with the potential to respond to growth factors. G0S19-1 is the main G0S19 gene expressed in adult T lymphocytes and may encode a homeostatic negative regulator of the size of cell populations (or subpopulations) which are derived ultimately from marrow stem cells. As such, it is a potential antioncogene.

Amino Acid Sequence↗
PubMed1992

Variable region sequence analysis of anti-DNA antibodies: evidence for a family of closely related germ-line VH genes encoding lupus autoantibodies.

Cloning and sequencing of the V regions of the anti-DNA monoclonal antibodies (mAbs), H438 and H130, indicate that H438 is encoded by a J558 VH gene, a single D region nucleotide, and unmutated JH1, V kappa-1C and J kappa 1 genes, and the H130 L chain is encoded by a V kappa-21 subgroup gene J kappa 1 gene. Identification of VH438, which shared VH hybridization pattern with 6% of a panel of 352 MRL/lpr hybridomas, suggests that the frequency of J558 use among spontaneously activated B cells in MRL/lpr mice is greater than previously reported. The VHH438 J558 family gene is identical to VHPAR, which encodes the independently derived MRL/lpr autoantibody, MRP-2, and is highly homologous to the previously reported VHH130, which is identical to a BALB/c germ-line VH gene. Comparison of consensus sequences of homologous autoantibodies and previously reported restriction mapping suggest that a minimum of three highly related J558 germ-line genes encode lupus autoantibodies.

Amino Acid Sequence↗
PubMed1994

Isolation of a cDNA encoding a metal response element binding protein using a novel expression cloning procedure: the one hybrid system.

A new method for isolation of cDNA clones encoding sequence-specific DNA-binding proteins is described. This method, the one-hybrid system, is based on the use of reporter genes whose transcription can be activated through synthetic cis elements recognized by the sought-after DNA-binding protein. These reporter genes are used for in vivo screening of a library of cDNAs fused to a DNA fragment encoding the GAL4 activation domain. cDNA clones expressing the appropriate fusion proteins lead to activation of these reporter genes in transformed yeast cells. We have used this approach to isolate a mammalian cDNA clone encoding a sequence-specific DNA-binding protein that recognizes the metal response elements (MREs) of the metallothionein (MT) genes. The protein encoded by this cDNA, M96, shows similarity to the trithorax proteins. Expression of a functional DNA-binding form of M96 requires Zn2+ ions. The recombinant protein binds to several different MREs but fails to recognize nonfunctional mutant MREs. M96 may be involved in the activation of MT genes in response to heavy-metal ions.

Amino Acid Sequence↗
PubMed1996

A human gene encoding diazepam-binding inhibitor/acy1-CoA-binding protein: transcription and hormonal regulation in the androgen-sensitive human prostatic adenocarcinoma cell line LNCaP.

Diazepam-binding inhibitor (DBI)/acyl-CoA-binding protein (ACBP) is a highly conserved 10-kD polypeptide expressed in various organs and implicated in the regulation of multiple biological processes such as GABAA/benzodiazepine receptor modulation, acyl-CoA metabolism, steroidogenesis, and insulin secretion. To extend our knowledge about the biology of DBI/ACBP and to elucidate the molecular mechanisms responsible for regulating DBI/ACBP gene expression, we have studied the androgen-regulated expression of DBI/ACBP transcripts in the human prostatic adenocarcinoma cell line LNCaP and have cloned and characterized a human gene encoding DBI/ACBP. Northern blotting, reverse transcription-assisted polymerase chain reaction (RT-PCR), ribonuclease protection, and 5' RACE analysis (rapid amplification of cDNA ends) of DBI/ACBP transcripts in LNCaP cells revealed androgen-regulated expression of multiple transcripts originating from multiple transcription start sites and alternative processing. The most abundant type of transcripts (referred to as type 1 transcripts) encodes genuine DBI/ACBP of 86 amino acids, while the minor type (type 2 transcripts) harbors an insertion of 86 bases and might encode an unrelated protein of 67 amino acids. Examination of a cloned DBI/ACBP gene revealed a structural organization of four exons present in all transcripts and one alternatively used exon present only in type 2 transcripts. The promoter region is located in a CpG island and lacks a canonical TATA box. Transient transfection of DBI/ACBP promoter fragments into LNCaP cells demonstrated that a region of 1.1 kb upstream of the translation start site is able to drive high-level expression of luciferase in LNCaP cells in an androgen-regulated fashion. Taken together these data indicate that the isolated human gene encoding DBI/ACBP is functional, has a high degree of structural similarity with the corresponding rat gene, exhibits hallmarks of a typical housekeeping gene, and harbors cis-acting elements that are at least partially responsible for androgen-regulated transcription in LNCaP cells.

Adenocarcinoma↗
PubMed2006

Isolation and characterization of cDNA encoding three dehydrins expressed during Coffea canephora (Robusta) grain development.

BACKGROUND AND AIMS: Dehydrins, or group 2 late embryogenic abundant proteins (LEA), are hydrophilic Gly-rich proteins that are induced in vegetative tissues in response to dehydration, elevated salt, and low temperature, in addition to being expressed during the late stages of seed maturation. With the aim of characterizing and studying genes involved in osmotic stress tolerance in coffee, several full-length cDNA-encoding dehydrins (CcDH1, CcDH2 and CcDH3) and an LEA protein (CcLEA1) from Coffea canephora (robusta) were isolated and characterized. METHODS: The protein sequences deduced from the full-length cDNA were analysed to classify each dehydrin/LEA gene product and RT-PCR was used to determine the expression pattern of all four genes during pericarp and grain development, and in several other tissues of C. arabica and C. canephora. Primer-assisted genome walking was used to isolate the promoter region of the grain specific dehydrin gene (CcDH2). KEY RESULTS: The CcDH1 and CcDH2 genes encode Y(3)SK(2) dehydrins and the CcDH3 gene encodes an SK(3) dehydrin. CcDH1 and CcDH2 are expressed during the final stages of arabica and robusta grain development, but only the CcDH1 transcripts are clearly detected in other tissues such as pericarp, leaves and flowers. CcDH3 transcripts are also found in developing arabica and robusta grain, in addition to being detected in pericarp, stem, leaves and flowers. CcLEA1 transcripts were only detected during a brief period of grain development. Finally, over 1 kb of genomic sequence potentially encoding the entire grain-specific promoter region of the CcDH2 gene was isolated and characterized. CONCLUSIONS: cDNA sequences for three dehydrins and one LEA protein have been obtained and the expression of the associated genes has been determined in various tissues of arabica and robusta coffees. Because induction of dehydrin gene expression is associated with osmotic stress in other plants, the dehydrin sequences presented here will facilitate future studies on the induction and control of the osmotic stress response in coffee. The unique expression pattern observed for CcLEA1, and the expression of a related gene in other plants, suggests that this gene may play an important role in the development of grain endosperm tissue. Genomic DNA containing the grain-specific CcDH2 promoter region has been cloned. Sequence analysis indicates that this promoter contains several putative regulatory sites implicated in the control of both seed- and osmotic stress-specific gene expression. Thus, the CcDH2 promoter is likely to be a useful tool for basic studies on the control of gene expression during both grain maturation and osmotic stress in coffee.

Amino Acid Sequence↗
PubMed2000

Adaptive encoding neural networks for the recognition of human signal peptide cleavage sites.

MOTIVATION: Data representation and encoding are essential for classification of protein sequences with artificial neural networks (ANN). Biophysical properties are appropriate for low dimensional encoding of protein sequence data. However, in general there is no a priori knowledge of the relevant properties for extraction of representative features. RESULTS: An adaptive encoding artificial neural network (ACN) for recognition of sequence patterns is described. In this approach parameters for sequence encoding are optimized within the same process as the weight vectors by an evolutionary algorithm. The method is applied to the prediction of signal peptide cleavage sites in human secretory proteins and compared with an established predictor for signal peptides. CONCLUSION: Knowledge of physico-chemical properties is not necessary for training an ACN. The advantage is a low dimensional data representation leading to computational efficiency, easy evaluation of the detected features, and high prediction accuracy. AVAILABILITY: A cleavage site prediction server is located at the Humboldt University http://itb.biologie.hu-berlin.de/ approximately jo/sig-cleave/ACNpredictor.cgi CONTACT: jo@itb.hu-berlin.de; berndj@zedat.fu-berlin.de

Binding Sites↗
PubMed2003

Neuroanatomical correlates of episodic encoding and retrieval in young and elderly subjects.

Lesion studies have shown convincingly that the medial temporal lobes (MTL) and frontal lobes are critical to episodic memory. Ageing generally has been found to have a generally negative effect on episodic memory performance, which might relate to neurofunctional changes in the frontal and medial temporal brain regions. In the present study, we used functional MRI (fMRI) to investigate separately the contributions of encoding and retrieval to the age-related decline in memory. To this end, we compared brain activity patterns obtained during incidental encoding (pleasant/unpleasant judgements about nouns) and subsequent retrieval (recognition) in three groups: a group of young subjects, a group of elderly subjects showing reduced memory performance (ELD-RED), and a group of elderly subjects who still performed in the normal range (ELD-NORM). This allowed us to differentiate between age-related changes in brain activity that affect memory function and those that do not have an apparent effect on memory function, because they are found in both elderly groups. Contrary to previous imaging studies on this topic, we used (self-paced) event-related fMRI to control for differences in performance level across groups by including correct responses only. Comparing the encoding of successfully remembered items with baseline (press left/press right), the young subjects showed a significant increase in brain activation in the left anterior MTL compared with the ELD-RED but not the ELD-NORM subjects. Comparing correctly rejected items (retrieval attempt) with baseline, the ELD-RED group showed much increased overall activity throughout the brain compared with the other groups. However, when correctly recognized items (retrieval attempt + success) were compared directly with correctly rejected items (retrieval attempt), these differences were greatly reduced, revealing common activity in the left parietal, retrosplenial and left anterior prefrontal regions. Therefore, we conclude that the reduced performance in the ELD-RED group is likely to be due to MTL dysfunction during encoding. The differences observed during retrieval attempts may reflect strategic differences. The lack of differences observed in relation to retrieval success suggests that ageing does not affect the processes that support the actual recovery of information.

Adult↗
PubMed2003

Age effects on the neural correlates of successful memory encoding.

Event-related functional MRI (fMRI) was used to investigate the neural correlates of memory encoding as a function of age. While fMRI data were obtained, 14 younger (mean age 21 years) and 14 older subjects (mean age 68 years) made animacy decisions about words. Recognition memory for these words was tested at two delays such that older subjects' performance at the short delay was comparable to that of the young subjects at the long delay. This allowed age-associated changes in the neural correlates of encoding to be dissociated from the correlates of differential recognition performance. Activity in left inferior prefrontal cortex and the left hippocampal formation was greater for subsequently recognized words in both age groups, consistent with the findings of previous studies in young adults. In the prefrontal cortex, these 'subsequent memory effects' were, however, left-lateralized in the younger group but bilateral in the older subjects. In addition, for the younger group only, greater activity for remembered words was observed in anterior inferior temporal cortex, as were reversed effects ('subsequent forgetting' effects) in anterior prefrontal regions. The data indicate that older subjects engage much of the same neural circuitry as younger subjects when encoding new memories. However, the findings also point to age-related differences in both prefrontal and temporal activity during successful episodic encoding.

Adolescent↗
PubMed2001

Task-dependency of the neural correlates of episodic encoding as measured by fMRI.

Using event-related functional magnetic resonance imaging (fMRI), the neural correlates of memory encoding can be studied by contrasting item-related activity elicited in a study task according to whether the items are remembered or forgotten in a subsequent memory test. Previous studies using this approach have implicated the left prefrontal cortex in the successful encoding of verbal material into episodic memory when the study task is semantic in nature. In the current study, we asked whether the neural correlates of episodic encoding differ depending on type of study task. Seventeen volunteers participated in an event-related fMRI experiment in which at study, volunteers were cued to make either animacy or syllable judgements about words. A recognition memory test followed after a delay of approximately 15 min. For the animacy task, words that were subsequently remembered showed greater activation in left and medial prefrontal regions. For the syllable task, by contrast, successful memory for words was associated with activations in bilateral intraparietal sulcus, bilateral fusiform gyrus, right prefrontal cortex and left superior occipital gyrus. These findings suggest that the brain networks supporting episodic encoding differ according to study task.

Adult↗
PubMed1998

Transmembrane-domain trapping: a novel method for isolation of cDNAs encoding putative membrane proteins.

We have developed a method that enables us to isolate cDNAs of putative membrane proteins. The system is designed to isolate a cDNA which can provide the transmembrane domain to the extracellular part of the IL-2 receptor alpha chain. We constructed a p18Mac vector by putting part of the IL-2 receptor alpha chain cDNA that encoded its signal sequence and extracellular domain, a cDNA cloning site and a poly(A) additional signal after a strong promoter SRalpha. If a cloned cDNA provides a transmembrane domain in-frame, the extracellular domain of the IL-2 receptor alpha chain will be expressed on the surface of the transfected cells. Otherwise, the chimeric protein will be either secreted or retained inside the transfected cells. We made a cDNA library using p18Mac and screened for cDNA clones which allowed the expression of the extracellular domain of the IL-2 receptor alpha chain on the cell surface. Of the 2000 clones screened, 5 clones were scored as positive. Partial sequence analysis revealed that one clone encoded the amyloid precursor protein, two others encoded mitochondrial proteins and the rest were new. These results suggest the system is effective in isolating cDNAs encoding putative membrane proteins.

Amino Acid Sequence↗
PubMed2002

Isolation of actin-encoding cDNAs from symbiotic corals.

A cDNA (named LGfact) encoding actin was identified in planular larvae of the scleractinian coral Galaxea fascicularis, using the reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques. RNA from the adult coral that was inhabited by symbiotic dinophytes was subjected to a similar RT-PCR, and a cDNA fragment, named AGfact-p, was found to encode an actin form distinct from LGfact. In an expression study, LGfact transcripts were present at similar levels in asymbiotic larvae and symbiotic adults, indicating that LGfact was expressed by the host. On the other hand, the expression of AGfact-p was detected in adults but not in larvae. Partial cDNA sequences of orthologues of LGfact and AGfact-p were detected in another scleractinian coral, Favites chinensis. A sequence identical to a part of AFcact-p (an AGfact-p orthologue) was amplified from the genomic DNA extracted from asymbiotic larvae of F. chinensis, strongly suggesting that AFcact-p was a coral actin cDNA. Thus, we presume that A Gfact-p encodes an adult-specific form of actin in the host. A partial actin-encoding cDNA sequence (named Syact-p) obtained from Symbiodinium sp. did not exhibit high levels of similarity to the coral actin sequences.

Actins↗
PubMed1996

The stoned locus of Drosophila melanogaster produces a dicistronic transcript and encodes two distinct polypeptides.

The stoned gene of Drosophila melanogaster is required for normal neuronal function in both adult and larva. We have identified DNA sequences that lie within a genetic region that is known to include the stoned gene and that also reveal restriction site variations in two stoned lethal mutants. This genomic region contains a single transcription unit coding for an approximately 8.4-kb transcript. The transcript is preferentially expressed in the head of adult flies. The isolation and sequencing of cDNA and genomic clones reveals that stoned appears to encode a dicistronic mRNA, although the possible existence of other forms of mRNA cannot be excluded. Antibody cross-reactivity shows that two proteins are translated from the stoned locus in vivo. Both open reading frames (ORFs) encode novel proteins. The protein encoded by the first ORF contains four tandemly repeated motifs, and one domain of the protein encoded by the second ORF shows similarity to a family of proteins (AP50s) associated with clathrin assembly protein complexes.

Amino Acid Sequence↗
PubMed2000

The Drosophila melanogaster ade5 gene encodes a bifunctional enzyme for two steps in the de novo purine synthesis pathway.

Steps 6 and 7 of de novo purine synthesis are performed by 5-aminoimidazole ribonucleotide carboxylase (AIRc) and 4-[(N-succinylamino)carbonyl]-5-aminoimidazole ribonucleotide synthetase (SAICARs), respectively. In vertebrates, a single gene encodes AIRc-SAICARs with domains homologous to Escherichia coli PurE and PurC. We have isolated an AIRc-SAICARs cDNA from Drosophila melanogaster via functional complementation with an E. coli purC purine auxotroph. This cDNA encodes AIRc yet is unable to complement an E. coli purE mutant, suggesting functional differences between Drosophila and E. coli AIRc. In vertebrates, the AIRc-SAICARs gene shares a promoter region with the gene encoding phosphoribosylamidotransferase, which performs the first step in de novo purine synthesis. In Drosophila, the AIRc-SAICARs gene maps to section 11B4-14 of the X chromosome, while the phosphoribosylamidotransferase gene (Prat) maps to chromosome 3; thus, the close linkage of these two genes is not conserved in flies. Three EMS-induced X-linked adenine auxotrophic mutations, ade4(1), ade5(1), and ade5(2), were isolated. Two gamma-radiation-induced (ade5(3) and ade5(4)) and three hybrid dysgenesis-induced (ade5(5), ade5(6), and ade5(8)) alleles were also isolated. Characterization of the auxotrophy and the finding that the hybrid dysgenesis-induced mutations all harbor P transposon sequences within the AIRc-SAICARs gene show that ade5 encodes AIRc-SAICARs.

Alleles↗
PubMed2001

The cyclope gene of Drosophila encodes a cytochrome c oxidase subunit VIc homolog.

Cytochrome c oxidase is the terminal enzyme of the mitochondrial electron transfer chain. In eukaryotes, the enzyme is composed of 3 mitochondrial DNA-encoded subunits and 7-10 (in mammals) nuclear DNA-encoded subunits. This enzyme has been extensively studied in mammals and yeast but, in Drosophila, very little is known and no mutant has been described so far. Here we report the genetic and molecular characterization of mutations in cyclope (cype) and the cloning of the gene encoding a cytochrome c oxidase subunit VIc homolog. cype is an essential gene whose mutations are lethal and show pleiotropic phenotypes. The 77-amino acid peptide encoded by cype is 46% identical and 59% similar to the human subunit (75 amino acids). The transcripts are expressed maternally and throughout development in localized regions. They are found predominantly in the central nervous system of the embryo; in the central region of imaginal discs; in the germarium, follicular, and nurse cells of the ovary; and in testis. A search in the Genome Annotation Database of Drosophila revealed the absence of subunit VIIb and the presence of 9 putative nuclear cytochrome c oxidase subunits with high identity scores when compared to the 10 human subunits.

Alleles↗
PubMed2002

Identification and characterization of the genes encoding the core histones and histone variants of Neurospora crassa.

We have identified and characterized the complete complement of genes encoding the core histones of Neurospora crassa. In addition to the previously identified pair of genes that encode histones H3 and H4 (hH3 and hH4-1), we identified a second histone H4 gene (hH4-2), a divergently transcribed pair of genes that encode H2A and H2B (hH2A and hH2B), a homolog of the F/Z family of H2A variants (hH2Az), a homolog of the H3 variant CSE4 from Saccharomyces cerevisiae (hH3v), and a highly diverged H4 variant (hH4v) not described in other species. The hH4-1 and hH4-2 genes, which are 96% identical in their coding regions and encode identical proteins, were inactivated independently. Strains with inactivating mutations in either gene were phenotypically wild type, in terms of growth rates and fertility, but the double mutants were inviable. As expected, we were unable to isolate null alleles of hH2A, hH2B, or hH3. The genomic arrangement of the histone and histone variant genes was determined. hH2Az and the hH3-hH4-1 gene pair are on LG IIR, with hH2Az centromere-proximal to hH3-hH4-1 and hH3 centromere-proximal to hH4-1. hH3v and hH4-2 are on LG IIIR with hH3v centromere-proximal to hH4-2. hH4v is on LG IVR and the hH2A-hH2B pair is located immediately right of the LG VII centromere, with hH2A centromere-proximal to hH2B. Except for the centromere-distal gene in the pairs, all of the histone genes are transcribed toward the centromere. Phylogenetic analysis of the N. crassa histone genes places them in the Euascomycota lineage. In contrast to the general case in eukaryotes, histone genes in euascomycetes are few in number and contain introns. This may be a reflection of the evolution of the RIP (repeat-induced point mutation) and MIP (methylation induced premeiotically) processes that detect sizable duplications and silence associated genes.

Amino Acid Sequence↗
PubMed1992

Transfection of HLA-DR-expressing DAP.3 cells with a cDNA clone encoding the glycosyl phosphatidylinositol-linked form of lymphocyte function associated antigen-3: biochemical features and functional consequences.

Structural and functional aspects of the accessory molecule lymphocyte function associated antigen (LFA)-3 (CD58) have been examined following the transfection of DAP.3 and P815 cells with a cDNA clone encoding the glycosyl phosphatidylinositol (GPI)-linked form of human LFA-3. Despite earlier observations that DAP.3 cells are deficient in GPI anchoring LFA-3 was expressed efficiently on DAP.3, as well as on P815 cells. Immunoprecipitation of LFA-3 from 35S-labelled cells revealed that the molecule expressed on the DAP.3 cells had a molecular weight intermediate between the transmembrane and GPI-linked forms expressed by human B cells. This suggests that the DAP.3 cells have a default pathway whereby the RNA transcript which encodes the GPI-linked form of the molecule can also encode an integral membrane protein. Functionally, expression of LFA-3 by DAP.3 which had previously been transfected with the genes encoding HLA-DR1 led to a marked augmentation of the proliferative response of five out of eight anti-DR1 human T cell clones. This effect was not reproduced when DR1 and LFA-3 were expressed by separate populations of DAP.3 cells, suggesting that the ligands for CD2 and for the T cell's receptor must be expressed on the same cell membrane. Expression of human LFA-3 also led to a substantial increase in the proliferative response of human peripheral blood T cells to a DR alloantigen. Separation of T cells into CD45RO+ and CD45RO- populations revealed that the augmentation was more marked for the memory than the virgin population. The mechanisms responsible for these differences are discussed.

Animals↗
PubMed1993

Molecular cloning of cDNAs encoding a LD78 receptor and putative leukocyte chemotactic peptide receptors.

Several cDNA clones encoding receptors for leukocyte chemoattractants, including IL-8, C5a, N-formyl peptides (FP), and platelet-activating factor, have been isolated in the past 3 years. The primary structure of these receptors revealed that they are members of the superfamily of G protein-coupled receptors containing seven transmembrane domains. In this study the polymerase chain reaction was carried out to isolate novel cDNA clones encoding human receptors of IL-8 related cytokines, chemokines, from a human monocyte cDNA library using degenerate oligonucleotide primers devised from conserved sequences among the cDNAs encoding the human receptors for IL-8, FP and C5a. Four novel cDNA clones (HM63, HM74, HM89, and HM145) in addition to cDNAs for FP and C5a receptors were isolated. All polypeptides encoded by the cloned cDNAs share common features with the G protein-coupled receptor superfamily, such as seven putative hydrophobic transmembrane domains and, except for HM74, N-linked glycosylation sites near the N-terminus. The amino acid sequence identities among HM63, HM89, HM145, IL-8 receptors, FP receptor, and C5a receptor are in the range of 24-68%, higher than those of other members of the G protein-coupled receptor superfamily. Moreover, the number of amino acids between the fifth and sixth transmembrane domains, which varies within this superfamily, is the same in these receptors. Thus, three of the newly identified proteins probably belong to a 'leukocyte chemotactic peptide receptor family'. HM74 differs from the other clones with respect to the amino acid homology, suggesting that this may be the receptor for a different type of ligand. Furthermore, it was confirmed that HM145 is a functional receptor for LD78, one of the C-C chemokines, as revealed by the measurement of decrease of cAMP accumulation as well as calcium influx using stable transfectants.

Amino Acid Sequence↗
PubMed1994

Identification of partial complementary DNA clones encoding a 59-kd protein with characteristics of a unique oncofetal antigen.

BACKGROUND: Oncofetal antigens (OFAs) are conserved tumor-associated autoantigens or transplantation antigens present on the surface of all major classes of rodent and human tumors and on midgestational fetal cells but not on normal neonatal or adult human and rodent tissues. A syngeneically derived monoclonal antibody, MAb-115, recognizes murine OFAs of 44 and 200 kd in molecular mass. PURPOSE: Our goal was to clone and characterize the complementary DNAs (cDNAs) that encode these murine OFAs. METHODS: Rabbit antiserum raised against purified 44-kd OFA glycoprotein was used to screen a mouse embryo cDNA-lambda phage expression library. Recombinant phage clones positive for the expression of OFAs were detected by immunohistochemical staining, then isolated and plaque purified. The presence of an OFA-encoding sequence in the recombinant phage was confirmed by specific reaction of the expressed protein with MAb-115. Recombinant fusion protein was purified from the extracts of corresponding lysogens. Rabbit antiserum against purified recombinant fusion protein was raised, and the capacity of this antiserum to detect the expression of OFA on rodent tumor and fetal cells was determined by flow cytometry. In addition, immunoreactivity of tumor bearer and hyperimmune murine sera to bacterially expressed recombinant OFA protein was evaluated by enzyme-linked immunosorbent assay. The OFA-expressing insert DNA from plaque-purified lambda clones was subcloned into phagemid vectors for sequencing analysis. RESULTS: Antiserum derived against the isolated recombinant mouse embryo polypeptide mimicked MAb-115 in its specific binding to all OFA-positive rodent tumor and fetal cell lines tested and likewise did not show reactivity to normal adult tissues. This antiserum specifically recognized the native 44- and 200-kd OFAs in extracts of murine lymphocytic lymphoma. Furthermore, sera of tumor-bearing mice or mice immunized with purified OFA or intact, irradiated OFA-positive lymphocytic lymphoma cells also reacted with the recombinant fusion protein. The characterization of the isolated clone included nucleotide sequence information followed by analysis of the deduced primary structure of the protein. CONCLUSIONS: These data suggest that the isolated cDNA clones encode a distinct gene product which is widely expressed on the surface of tumor and fetal cells and represents the first characterized sequence of a true OFA. IMPLICATIONS: The availability of this cDNA, encoding a protein expressed only on tumor and fetal cells, provides a direct means to assess biological characteristics of malignant tissue which can be assayed by biochemical, histochemical, and molecular methods.

Amino Acid Sequence↗