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PubMed1991

Evidence for a senescence-associated gene induced by darkness.

A nearly full-length cDNA was isolated from a cDNA library prepared from incipiently senescent radish (Raphanus sativus L.) cotyledons using a previously isolated cDNA clone for dark-inducible mRNA as a probe (A Watanabe, N Kawakami, Y Azumi [1989] In Cell Separation in Plants, NATO ASI Series, Vol H35, pp 31-38. Springer-Verlag, Berlin). The clone detected transcripts of 800 bases which increased more than 100-fold after 24 hours of darkness. The transcripts also accumulated under light when plants were exposed to ethylene or heat stress, and (6)N-benzyladenine partially repressed its accumulation in the dark. These responses of the gene to physiological stimuli closely paralleled the effects of the stimuli on the progress of senescence of the cotyledons. We have named the gene din 1 (dark inducible gene 1). The cDNA encodes a polypeptide of 20 kilodaltons, and its nucleotide sequence shows a high (49%) similarity to a subfamily of pathogenesis-related proteins of tobacco. The predicted amino acid sequence of the product, however, shows only 20% homology to the pathogenesis-related protein.

Journal Article↗

PubMed1991

Primary structure of sweet potato starch phosphorylase deduced from its cDNA sequence.

Sweet potato (Ipomoea batatas) starch phosphorylase cDNA clones were isolated by screening an expression library prepared from the young root poly(A)(+) RNA successively with an antiserum, a monoclonal antibody, and a specific oligonucleotide probe. One cDNA clone had 3292 nucleotide residues in which was contained an open reading frame coding for 955 amino acids. This sequence was compared with those of potato (916 residues plus 50-residue putative transit peptide) and rabbit muscle (841 residues) phosphorylases. The sweet potato phosphorylase has an overall structural feature highly homologous to that reported for potato phosphorylase, in conformity with the finding that they belong to the same class of plant phosphorylase. High divergencies of the two enzymes are found in the about 70 residue N-termini each including a putative transit peptide, and the midchain 78 residue insert typical of type I plant phosphorylase. We consider that the very high dissimilarity found in the midchain inserts is related to the difference in proteolytic lability of the two plant phosphorylases. Some structural features of the cDNA clone were also discussed.

Journal Article↗

PubMed2003

Genes encoding proteins of the cation diffusion facilitator family that confer manganese tolerance.

The yeast Saccharomyces cerevisiae expressing a cDNA library prepared from Stylosanthes hamata was screened for enhanced Mn(2+) tolerance. From this screen, we identified four related cDNAs that encode membrane-bound proteins of the cation diffusion facilitator (CDF) family. One of these cDNAs (ShMTP1) was investigated in detail and found to confer Mn(2+) tolerance to yeast by internal sequestration rather than by efflux of Mn(2+). Expression of ShMTP1 in a range of yeast mutants suggested that it functions as a proton:Mn(2+) antiporter on the membrane of an internal organelle. Similarly, when expressed in Arabidopsis, ShMTP1 conferred Mn(2+) tolerance through internal sequestration. The ShMTP1 protein fused to green fluorescent protein was localized to the tonoplast of Arabidopsis cells but appeared to localize to the endoplasmic reticulum of yeast. We suggest that the ShMTP1 proteins are members of the CDF family involved in conferring Mn(2+) tolerance and that at least one of these proteins (ShMTP1) confers tolerance by sequestering Mn(2+) into internal organelles.

Adaptation, Physiological↗

PubMed1991

Expression patterns of myb genes from Antirrhinum flowers.

Six genes that contain sequence encoding the DNA binding domain of the Myb oncoproteins have been isolated from a cDNA library prepared from Antirrhinum majus (snapdragon) flowers using oligonucleotide probes directed against part of this domain. The derived amino acid sequences of these genes reveal acidic domains in their carboxy termini, suggesting that they might act as transcriptional activators. Analysis of their expression patterns with respect to organ specificity, floral differentiation, and response to light suggests that these genes are not involved in controlling anthocyanin biosynthesis, unlike the characterized myb-related genes C1 and Pl from maize. One of the genes is expressed mainly in the nectary and the transmitting tract of the style, two major secretory tissues of the flower, suggesting that the function of this gene is related to active carbohydrate secretion. We conclude that plants contain a number of myb-related transcriptional activators involved in a diversity of gene regulation.

Amino Acid Sequence↗

PubMed1992

Epidermis-specific gene expression in Pachyphytum.

Transcripts of exclusively epidermis-specific expression were obtained by differential screening of a cDNA library prepared from isolated epidermis tissue of a succulent plant, Pachyphytum. Six transcripts were selected and characterized by RNA gel blot hybridization. Five cDNAs represented abundant mRNAs found exclusively in the epidermis. In situ hybridizations with three of these transcripts further substantiated their epidermal location. While two transcripts were present in all cells of the epidermis, one transcript was enriched in subsidiary cells of the stomatal complexes. All of the transcripts were highly expressed in the protoderm of the shoot apical meristem. DNA sequence determination indicated that the mRNAs identified represent novel sequences, encoding yet unknown plant functions. These transcripts, their control elements, and their encoded functions should help to advance our understanding of epidermal cell determination and differentiation.

Amino Acid Sequence↗

PubMed1992

Specific expression of an extensin-like gene in the style of Nicotiana alata.

cDNAs and corresponding genomic clones encoding a putative proline-rich protein (NaPRP3) were isolated from libraries prepared from Nicotiana alata style mRNA and genomic DNA. The predicted NaPRP3 protein is structurally similar to extensin in containing six copies of the characteristic extensin sequence Ser-Pro4, but differs in being smaller (151 residues compared with greater than 300 residues) and lacking Tyr residues. In contrast to most extensin genes, the NaPRP3 gene is not induced by mechanical wounding, and its expression is restricted to cells of the transmitting tract of the style.

Amino Acids↗

PubMed1993

An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence.

An Arabidopsis cDNA (Atmyb2) that contains a sequence that encodes a transcription factor, which is a homolog of MYB, was cloned from a cDNA library prepared from dehydrated Arabidopsis rosette plants. A gene (Atmyb2) corresponding to the Atmyb2 cDNA was also cloned and its nucleotide sequence was determined. RNA gel blot analysis showed that the Atmyb2 mRNA was induced by dehydration and disappeared upon rehydration. The Atmyb2 mRNA also accumulated upon salt stress and with the onset of treatment with abscisic acid. A beta-glucuronidase reporter gene driven by the Atmyb2 promoter was induced by dehydration and salt stress in transgenic Arabidopsis plants. These observations indicate that Atmyb2 is responsive to dehydration at the transcriptional level. The putative protein (ATMYB2) encoded by Atmyb2 has 274 amino acids, a molecular mass of 32 kD, and a putative DNA binding domain that shows considerable homology to plant MYB-related proteins, such as maize C1. A fusion protein that included ATMYB2 was expressed in Escherichia coli, and it bound specifically to oligonucleotides that contained a consensus MYB recognition sequence (TAACTG), such as is found in the simian virus 40 enhancer and the maize bronze-1 promoter. Binding was sequence specific, as indicated by a gel mobility shift experiment. These results suggest that a MYB-related transcription factor is involved in the regulation of genes that are responsive to water stress in Arabidopsis.

Abscisic Acid↗

PubMed1993

Molecular characterization of NADH-dependent glutamate synthase from alfalfa nodules.

Alfalfa NADH-dependent glutamate synthase (NADH-GOGAT), together with glutamine synthetase, plays a central role in the assimilation of symbiotically fixed nitrogen into amino acids in root nodules. Antibodies previously raised against purified NADH-GOGAT were employed to screen a cDNA library prepared using RNA isolated from nodules of 20-day-old alfalfa plants. A 7.2-kb cDNA clone was obtained that contained the entire protein coding region of NADH-GOGAT. Analysis of this cDNA and determination of the amino-terminal amino acids of the purified protein revealed that NADH-GOGAT is synthesized as a 2194-amino acid protein that includes a 101-amino acid presequence. The deduced amino acid sequence shares significant identity with maize ferredoxin-dependent GOGAT, and with both large and small subunits of Escherichia coli NADPH-GOGAT. DNA gel blot analysis of alfalfa genomic DNA suggests the presence of a single NADH-GOGAT gene or a small gene family. The expression of NADH-GOGAT mRNA, enzyme protein, and enzyme activity was developmentally regulated in root nodules. A dramatic increase in gene expression occurred coincidentally with the onset of nitrogen fixation in the bacteroid, and was absent in both ineffective plants that were nodulated with effective Rhizobium meliloti and effective plants that had been nodulated with ineffective R. meliloti strains. Maximum NADH-GOGAT expression, therefore, appears to require an effective, nitrogen-fixing symbiosis.

Amino Acid Sequence↗

PubMed1999

Characterization of the Coxiella burnetti sucB gene encoding an immunogenic dihydrolipoamide succinyltransferase.

The Coxiella burnetii sucB gene encoding the dihydrolipoamide succinyltransferase (E2o) enzyme was cloned by immunological screening of a lambda EMBL3 genomic library prepared from strain Nine Mile DNA and sequenced. The homology of the cloned gene product to the counterpart in Escherichia coli was 54.3%, but the homology of the N-terminal region was only 42%. The gene was expressed in E. coli as an independent unit from its own promoter, producing an immunoreactive protein of about 50 kDa on SDS-PAGE which reacted with antisera from laboratory animals and sera from human patients with acute Q fever. The study results suggest that the C. burnetii E2o enzyme may serve as a potential target antigen for diagnostic assays for Q fever.

Acyltransferases↗

PubMed1995

Isolation of a cDNA for a growth factor of vascular endothelial cells from human lung cancer cells: its identity with insulin-like growth factor II.

We have found growth-promoting activity for vascular endothelial cells in the conditioned medium of a human lung cancer cell line, T3M-11. Purification and characterization of the growth-promoting activity have been carried out using ammonium sulfate precipitation and gel-exclusion chromatography. The activity migrated as a single peak just after ribonuclease. It did not bind to a heparin affinity column. These results suggest that the activity is not a heparin-binding growth factor (including fibroblast growth factors) or a vascular endothelial growth factor. To identify the molecule exhibiting the growth-promoting activity, a cDNA encoding the growth factor was isolated through functional expression cloning in COS-1 cells from a cDNA library prepared from T3M-11 cells. The nucleotide sequence encoded by the cDNA proved to be identical with that of insulin-like growth factor II.

Animals↗

PubMed2001

Detection and cloning of a protein recognized by anti-human prostate-specific antigen (PSA) antibody in the rat ventral prostate.

Prostate-specific antigen (PSA), a 33 kDa glycoprotein produced in the epithelium of the human prostate, has become established as a useful tumor marker for prostate cancer in man. Since reports of homologous proteins in animals other than primates have been lacking, the present investigation was carried out to identify any PSA-like protein in rats. Immunoblot analysis using a specific monoclonal anti-human PSA antibody detected a 32 kDa immunoreactive protein in the ventral lobe of the rat prostate, but not in other lobes or in other tissues. Positive immunostaining was observed only for the luminal surface of the glandular epithelium and the intraductal fluid in the ventral prostate. Sequence analysis of a cDNA for the rat PSA-like protein, cloned by immunoscreening of an expression cDNA library prepared from the ventral lobe, revealed identity to the rat submaxillary gland S3 kallikrein. Human PSA also belongs to the kallikrein family. Thus, this protein produced in the rat ventral prostate was suggested to be a possible counterpart of human PSA.

Amino Acid Sequence↗

PubMed2006

Heat shock protein 105 is overexpressed in squamous cell carcinoma and extramammary Paget disease but not in basal cell carcinoma.

BACKGROUND: Heat shock protein (HSP) 105 is a 105-kDa protein, recently discovered by serological analysis of recombinant cDNA expression libraries prepared from tumour cells (SEREX), and is still undergoing intensive research. SEREX can define strongly immunogenic tumour antigens that elicit both cellular and humoral immunity. Previous studies have shown that HSP105 is a cancer testis antigen and is overexpressed in various internal malignancies. The expression of HSP105 has not been studied in skin cancers. OBJECTIVES: To assess the expression of HSP105 in skin cancers including extramammary Paget disease (EMPD), cutaneous squamous cell carcinoma (SCC) and basal cell carcinoma (BCC). METHODS: Samples of EMPD (n = 25), SCC (n = 23, of which three were metastatic lesions) and BCC (n = 23) were collected from patients treated in our department between January 2002 and December 2004. Western blot and immunohistochemical staining methods were used to investigate the expression of HSP105. RESULTS: Results of Western blot analysis showed overexpression of HSP105 in EMPD and SCC, and minimal expression in BCC. Immunohistochemistry results showed that 56% of EMPD, 60% of primary and 100% of metastatic SCC highly expressed HSP105 while only 13% of BCC lesions showed increased staining. CONCLUSIONS: EMPD and SCC overexpress HSP105 while BCC does not. Tumours overexpressing HSP105 present ideal candidates for vaccination by HSP105-derived peptides or DNA.

Aged↗

PubMed1997

Immunological approach to hepatocellular carcinoma.

A library of monoclonal antibodies (MoAbs) has been produced against a human hepatocellular carcinoma (HCC) cell line designated FOCUS in order to study the antigenic properties of transformed hepatocytes. Several monoclonal antibodies (MoAbs) were initially selected for study since they bound to antigens which were overexpressed in HCC tissues compared with the adjacent uninvolved normal liver counterpart; in addition, these MoAbs revealed low level antigen expression on other normal human tissues. Subsequently, HCC cell lines were metabolically labelled and the antigens further characterized by immunoprecipitation and Western blot analysis. If the MoAb recognized a primary linear epitope on a protein, cloning was performed using a lambda GT11 cDNA expression library prepared from the FOCUS HCC cell line. These studies characterized the HCC associated antigen(s) at the molecular level. This review illustrates the value of such an experimental approach to search for and identify HCC associated antigens and emphasizes the biological properties of novel proteins may be defined and characterized by these techniques. More important, our investigations have described unique proteins that may not only be important in the pathogenesis of HCC but also demonstrates how such antigen-antibody systems may be used to develop strategies for immunotargetting and gene therapy of HCC.

Animals↗

PubMed1991

Identification of a novel S-phase-specific gene during the cell cycle in synchronous cultures of Catharanthus roseus cells.

The cell-cycle specific cDNAs were isolated from a cDNA library prepared from cells in the S phase in the synchronous cultures of Catharanthus roseus. One of the isolated genes, which we refer to as cyc07, was analyzed in detail. The full-length cDNA of cyc07 contains an open reading frame of 735 nucleotides, encoding a protein of 245 amino acids with a molecular weight of 28,356 Da. The protein predicted from the nucleotide sequence is highly basic, as are mammalian histones. cyc07 mRNA was detected specifically in cells at the S phase in synchronous cultures. The induction and accumulation of mRNA in the S phase were suppressed when DNA synthesis was inhibited by aphidicolin. In the intact plant, cyc07 mRNA was found preferentially in root tips that contained meristematic tissue. A databank search revealed that a sequence homologous to the nucleotide sequence of cyc07 cDNA is present in the downstream region of the SIR3 gene in the yeast genome. The amino acid sequence predicted from the corresponding region of the yeast genome exhibited significant homology with that of cyc07 protein. These similarities between cyc07 and the corresponding region in yeast suggest that the homologous sequence in yeast is a novel gene that is functionally homologous to cyc07. Our results presented here suggest the possibility that cyc07 may play a role in the proliferation of higher plant cells, in particular in the entry into or progression of the S phase of the cell cycle.

Amino Acid Sequence↗

PubMed1986

Characterization of cloned sequences complementary to F9 cell double-stranded RNA and their expression during differentiation.

In a cDNA library prepared from the RNA of cultured murine F9 teratocarcinoma cells, we identified sequences exhibiting strong hybridization to double-stranded RNA (dsRNA) of F9 cells but weak hybridization to mouse liver dsRNA. Northern-blot hybridization of RNA extracted from F9 cells with or without treatment with retinoic acid revealed differences in the expression of some of these sequences in undifferentiated and differentiated cells. As shown by Southern-blot hybridization experiments, these differences of expression were not related to a gross rearrangement of the corresponding genomic DNA sequences of the differentiated cells. When RNA from F9 cells was used, one of the cloned dsRNA-related sequences selected mRNA which was translated in vitro to a polypeptide with an Mr of 24,000; the level of this mRNA was reduced in F9 cells that had been treated with retinoic acid. Our results show that the differentiation of F9 cells induced by retinoic acid results in the differential expression of some middle-repetitive sequences.

Animals↗

PubMed1986

Cytokeratin expression in simple epithelia. II. cDNA cloning and sequence characteristics of bovine cytokeratin A (no. 8).

Cytokeratin A (no. 8) is a cytoskeletal protein (Mr, approximately 53,000 in bovine cells) which is typical of all simple epithelia, is widespread in all cultured epithelial cells, and together with its partner cytokeratin D, is the first cytokeratin expressed during embryogenesis (synonyms for this protein are Endo A and TROMA-1 antigen). We isolated a clone (pKB8(1] from a pUC8 cDNA library prepared from poly(A)+-RNA of bovine bladder urothelium which contains the 3' nontranslated portion and the sequence coding for the carboxyterminal tail and almost the whole of the alpha-helical rod (369 amino acids). Northern-blot analysis showed that the mRNA coding for this cytokeratin is specifically synthesized in various epithelial tissues and in epithelial cell culture lines. The amino acid sequence of this cytokeratin, when compared with the sequences of other intermediate filament (IF) proteins, exhibits a high and specific homology with other cytokeratins of the basic (type II) subfamily; this homology is, however, restricted to the rod portion. The tail region, which is rich in hydroxy-amino acids (approximately 35%), is unique among the type-II cytokeratins in that it does not exhibit subdivision in three domains, specifically lacking the glycine-rich middle domain. Sequence comparison with a partial sequence of the corresponding cytokeratin of the amphibian species, Xenopus laevis, indicated high evolutionary conservation. The high sequence homology of bovine cytokeratin A with published sequences of human tissue polypeptide antigen (TPA), a soluble serum component used as tumor marker in clinical oncology, supports the view that TPA is a proteolytically solubilized fragment containing the rod portion of human cytokeratin no. 8. Our analysis of clone pKB8(1) made possible the first comparison of a simple epithelial cytokeratin with epidermal keratins and other IF proteins. This showed that, in some important molecular features, cytokeratin A (no. 8) differs drastically from the epidermal members of the same cytokeratin subfamily, probably reflecting different cellular functions of the tail region in stratified and simple epithelia.

Amino Acid Sequence↗

PubMed1988

Sequence analysis of murine cytokeratin endo A (no. 8) cDNA. Evidence for mRNA species initiated upstream of the normal 5' end in PCC4 cells.

Keratin 8 (Endo A) is expressed in simple epithelia, together with keratin 18 (Endo B). Filaments formed by this keratin pair are the first cytoskeletal elements induced during mouse embryogenesis. We have isolated Endo A cDNA clones from lambda gt11 libraries prepared with mRNA isolated from PCC4 embryonal carcinoma (EC) cells. Sequencing of three overlapping cDNAs and of a genomic clone allowed us to determine the complete sequence of the Endo A message. Analysis of the protein sequence deduced showed that the Endo A protein presented all the characteristics of intermediate filaments, including an alpha-helical central rod domain and nonhelical N- and C-termini. In the rod domain, the degree of similarity to the other members of the basic keratin family was high. A high degree of homology to keratin 8 of other species was observed, even in the non-helical domains. During these analyses, we found clones extending upstream of the normal 5' end of the mRNA. Sequence comparison between these cDNAs and the 5' upstream region of the Endo A gene suggested that they corresponded to transcripts initiated at an upstream alternative promoter. These observations supported previous results showing the presence of Endo A transcripts initiated upstream of the normal 5' end in mouse morulae and blastocysts.

Amino Acid Sequence↗

PubMed1985

Cloning, nucleotide sequencing and expression of cDNAs encoding mouse urokinase-type plasminogen activator.

Controlled extracellular proteolysis is catalyzed in part by the secretion of plasminogen activators. As a step in the study of the expression of these enzymes in mouse tissues, we have isolated five cDNAs encoding the mouse urokinase-type plasminogen activator from a cDNA library prepared with size-selected mRNA from MSV-transformed 3T3 cells. The longest cDNA insert contains the entire coding region of mouse urokinase, 58 base pairs of the 5' non-coding region, and the entire 3' non-coding region, which is 942 base pairs long. The deduced protein sequence, which starts with a signal peptide of 20 amino acids, shows extensive homology to that of human and porcine urokinase. However, in contrast to these enzymes, mouse urokinase contains no N-glycosylation site. Bacteria harbouring one of the recombinant plasmids synthesize and secrete into their periplasm a protease indistinguishable from mouse urokinase.

Amino Acid Sequence↗