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At least 883 records · Page 49Linked to original sources
PubMed1994

Cloning and characterization of OSF-3, a new member of the MER5 family, expressed in mouse osteoblastic cells.

A cDNA library prepared from the mouse osteoblastic cell line, MC3T3-E1, was screened for the presence of specifically expressed genes by employing subtraction hybridization/differential screening methods. A cDNA clone was identified and sequenced, encoding a protein designated as osteoblast specific factor 3 (OSF-3) comprising 199 amino acids. RNA dot blot analysis indicated weak OSF-3 expression in thymus, spleen, brain, lung, testis, and heart, and high expression in kidney and liver. A homology search of an amino acid sequence database revealed a strong relationship of OSF-3 to the MER5 (gene preferentially expression in murine erythroleukemia cells) protein and human pag (proliferation associated gene) product. This indicates that OSF-3 plays an intrinsic role in the proliferation and/or differentiation of bone cells.

Amino Acid Sequence↗
PubMed1996

cDNA cloning and expression of a gene for isocitrate lyase in pumpkin cotyledons.

A cDNA clone for isocitrate lyase (ICL) was isolated from a cDNA library prepared from the poly(A)+ RNA of etiolated pumpkin cotyledons. The cDNA encoded a polypeptide with 576 amino acids, whose sequence is more than 79% identical to those of ICL from other higher plants and contains the C-terminal tripeptide, Ser-Arg-Met, which is a putative targeting signal to microbodies. Immunogold analysis revealed that ICL protein is exclusively localized in microbodies in etiolated pumpkin cotyledons. Double labeling experiments with protein A-gold particles of different sizes showed that ICL protein coexists with a leaf-peroxisomal enzyme, glycolate oxidase, in the microbodies of greening and senescing pumpkin cotyledons, indicating that transformation between glyoxysomes and leaf peroxisomes occurs directly during greening and senescence of the cotyledons. Immunoblot analysis showed that the amount of ICL protein increased markedly during germination and decreased rapidly when seedlings were exposed to illumination, which induced the microbody transition from glyoxysomes to leaf peroxisomes. In senescing cotyledons, the level of the ICL protein and ICL mRNA increased again with the reverse transition of microbodies from leaf peroxisomes to glyoxysomes. Changes in the amount of ICL protein did not correspond to the changes in the level of ICL mRNA during greening and senescence of the cotyledons, an indication that post-transcriptional regulation plays an important role in the microbody transition.

Amino Acid Sequence↗
PubMed1997

Gene expression during cold acclimation in strawberry.

To elucidate the molecular basis of cold acclimation in strawberry (Fragaria x anannassa), we have begun studies to identify genes associated with low temperature (LT) acclimation. Differential screening of a cDNA library prepared from cold-acclimated strawberry plants allowed us to isolate several cDNAs showing differential expression at LT. Northern analysis showed that the transcript level of Fcor1 (Fragaria Cold-Regulated) peaked after 2 days of LT exposure while that of Fcor2 peaked after 2 weeks. On the other hand, the level of Fcor3 transcript decreased within 24 hours of LT exposure and remained low during the 8 weeks acclimation period. Fcor1 and Fcor2 are expressed in all tissues while Fcor3 is specific to leaves. The Fcor1-encoded protein has a compositional bias for leucine, isoleucine, glycine, proline and serine. This protein shares homology with the proteins encoded by blt101, a LT-responsive gene from barley, and ESI3, a gene induced by salt stress in Lophopyrum. The FCOR2 protein is rich in lysine, leucine, valine, alanine and arginine, and shows no homology with any known gene products. The partial Fcor3 cDNA clone encodes a polypeptide that shows a very high identity with the spinach PSI subunit V and with the PSI PsaG polypeptide from barley. The level of Fcor1 transcript accumulation is correlated with the freezing tolerance of the strawberry cultivars used in our study. This suggests that Fcor1 may be useful as a molecular marker to select for this trait in resulted species of the Rosaceae family.

Acclimatization↗
PubMed1998

Molecular cloning and expression of the early auxin-responsive Aux/IAA gene family in Nicotiana tabacum.

Early auxin-regulated tobacco cDNAs, belonging to the Aux/IAA gene family have been isolated by screening of a cDNA library prepared from auxin-treated suspension-grown etiolated seedlings of Nicotiana tabacum. The probes used were either RT-PCR fragments or an insert resulting from mRNA differential display selection. All of them possessed the structural features which characterize the Aux/IAA gene products. The auxin response of three distinct Nt-iaa subclasses has been characterized in terms of kinetics, dose-response and specificity as several plant hormones and chemicals have been tested for their ability to alter Nt-iaa mRNA accumulation. Differences of auxin responses have been observed between the Nt-iaa analysed, revealing significant differences of regulation. The effect of the protein synthesis inhibitor cycloheximide suggested that Nt-iaa2.3, Nt-iaa4.3 and strictly related genes can be classified as primary auxin-responsive genes and Nt-iaa28 as a late one. The steady-state mRNA level of these Nt-iaa has also been determined in organs of tobacco plants.

Amino Acid Sequence↗
PubMed1998

Root-specific expression of genes for novel glycine-rich proteins cloned by use of an antiserum against xylem sap proteins of cucumber.

Candidate cDNAs for xylem sap proteins (XSPs) were isolated by screening of a cucumber cDNA library prepared from poly(A)+ RNA of cucumber roots with an antiserum raised against the XSPs of cucumber. The patterns of expression of corresponding transcripts and the sequences of the cDNAs were examined. Two cDNAs with a glycine-rich domain in each deduced amino acid sequence and whose transcripts were expressed predominantly in roots were selected for further analysis. Both cDNAs encoded proteins with a putative signal sequence, with similar non-glycine-rich domains at the amino terminal of the encoded polypeptides. The corresponding mRNAs accumulated concurrently with the formation of adventitious roots from cuttings of cucumber hypocotyls and the development of vascular tissues, and strong expression of the mRNAs was detected in the root-hair zone of tap roots. These data suggest that the production of the two novel glycine-rich proteins, which are putative XSPs, might be associated with the functions of the vascular tissues in roots.

Amino Acid Sequence↗
PubMed1995

Molecular cloning and characterization of cDNA for a rice protein that contains seven repetitive segments of the Trp-Asp forty-amino-acid repeat (WD-40 repeat).

A cDNA clone for a polypeptide that contained seven repetitive segments of the Trp-Asp forty-amino-acid repeat (WD-40 repeat) was isolated from a cDNA library prepared from the greening leaves of rice. The cDNA was 1,285 bp long and contained an open reading frame that encoded a protein of 334 amino acid residues, which was designated it RWD (rice protein containing the WD-40 repeat). RWD exhibited greater homology to a group of receptor for activated C-kinase (RACK), a product of auxin-regulated gene from cultured cells (arcA) and a Chlamydomonas beta subunit-like polypeptide (Cblp) rather than to the beta subunits of heterotrimeric G protein complexes. The mRNA for RWD (1.3 kb) was found in all organs of rice plants, in particular, in roots. Therefore, RWD is suggested to be a protein that is expressed constitutively.

Amino Acid Sequence↗
PubMed1995

Cloning of a carrot cDNA for a member of the family of ADP-ribosylation factors (ARFs) and characterization of the binding of nucleotides by its product after expression in E. coli.

A cDNA clone, designated DcArf1, was isolated from a cDNA library prepared from embryogenic cell clusters of Daucus carota and characterized. The cDNA (883 bp) contained an open reading frame that encoded a protein of 181 amino acid residues with significant homology to the ADP-ribosylation factors (ARFs) of animals and yeast. The DcArf1-related transcripts were detected at a nearly constant level during somatic embryogenesis. The DcArf1-encoded protein was expressed as a fusion protein in E. coli and was shown to have dithiothreitol-independent [alpha-32P]GTP-binding activity. This binding was completely prevented by a 100-fold molar excess of unlabeled GTP or GDP, while GMP, ANP, TNP, CNP and UNP had no effect on the binding. The binding of [35S]GTP gamma S to the recombinant DCARF1 protein was detected only in the presence of millimolar levels of both MgCl2 and dimyristoylphosphatidylcholine (DMPC). The amino acid sequence and the GTP-binding characteristics of the DCARF1 protein suggest that the DcArf1 cDNA encodes an ARF of carrot rather than a protein that only resembles such a factor, ADP-ribosylation factor-like protein (ARL).

ADP-Ribosylation Factors↗
PubMed2000

Expression of two uricase (Nodulin-35 genes in a non-ureide type legume, Medicago sativa.

Two cDNA clones, MsU2 and MsU9 encoding uricase (EC 1.7.3.3, Nodulin-35) were isolated from a cDNA library prepared from nodule tissues of alfalfa, Medicago sativa, plants. Both MsU2 and MsU9 encoded 308 amino acid polypeptides with a difference of 5 amino acids, and the deduced amino acid sequences shared 98% homology. Between these two cDNA clones and uricase genes of soybean which were designated as Nod-35s, more than 80% identity was observed in nucleotides and deduced amino acid sequences, suggesting that these MsU2 and MsU9 are homologs of Nod-35. Using the reverse transcription-PCR technique, we detected the transcripts of these two genes in almost all tissues of alfalfa. The operation of uricase genes was confirmed by the presence of ureide in the xylem sap and uricase activity in the nodules. In situ hybridization analysis revealed that MsU2 and MsU9 were expressed only in uninfected cells of the infected zone of the nodule tissue. The cell specific-expression of the two uricase genes was observed in an identical manner to that of Nod-35 in soybean nodules.

Amino Acid Sequence↗
PubMed2004

Cloning and characterization of glycine-rich RNA-binding protein cDNAs in the moss Physcomitrella patens.

We isolated three cDNAs for the genes PpGRP1, PpGRP2 and PpGRP3 that encode glycine-rich RNA-binding proteins (GRPs) from Physcomitrella patens. Three full-length cDNA clones were isolated from a cDNA library prepared from poly(A)(+) RNA from 7-day-old protonemata of P. patens. They were named PpGRP1, PpGRP2 and PpGRP3, which encode putative polypeptides of 162, 178 and 155 residues, respectively. Preliminary genomic sequencing suggested that the positions of the three introns in the PpGRP3 gene are similar to those of introns in Arabidopsis GRP genes. PpGRP3 had a putative transit sequence. The PpGRP1-sGFP and PpGRP2-sGFP fusions were targeted to the cell nucleus, while PpGRP3-sGFP fusion was targeted to mitochondria. The level of these PpGRP transcripts as well as that of PpGRP proteins increased after cold treatment. Homoribopolymer RNA assay revealed that PpGRP3 protein show high affinity for poly(U) and poly(G). Results of phylogenetic analysis suggest that the nuclear and mitochondrial forms of GRP have been established early during the evolution of green plants.

Amino Acid Sequence↗
PubMedDate not supplied

Resistance to tomato spotted wilt virus infection in transgenic tobacco expressing the viral nucleocapsid gene.

A recombinant plasmid containing the entire tomato spotted with virus (TSWV) nucleocapsid gene, with the exception of nucleotide encoding three N-terminal amino acids, was isolated by screening a complementary DNA library, prepared against random primed viral RNA, using a specific monoclonal antibody. The insert contained in plasmid pTSW1 was repaired and amplified by polymerase chain reaction, and the complete nucleocapsid protein gene was introduced into Nicotiana tabacum 'Samsun' by leaf disk transformation using Agrobacterium tumefaciens. Transgenic plants expressing the viral nucleocapsid protein were resistant to subsequent infection following mechanical inoculation with TSWV as indicated by a lack of systemic symptoms and little or no systemic accumulation of virus as determined by double antibody sandwich enzyme-liked immunosorbent assay. These results further extend the applicability of coat protein-mediated resistance, as previously demonstrated for a number of simple plant viruses composed of a positive-sense RNA genome encapsidated with a single species of coat protein, to a membrane-encapsidated, multi-component, negative-sense RNA virus.

Base Sequence↗
PubMedDate not supplied

Purification, characterization, and molecular cloning of basic PR-1-type pathogenesis-related proteins from barley.

Partial amino acid sequences of two proteins, purified from barley leaves reacting hypersensitively to the powdery mildew fungus, showed a high degree of amino acid identity to the PR-1 proteins originally described in tobacco. The proteins, subsequently designated HvPR-1a and HvPR-1b, show apparent pI values of approximately 10.5 and 11, respectively and apparent M(r) 15,000. Independently, differential screening of a cDNA library prepared from barley leaves, exhibiting a compatible interaction with the powdery mildew fungus, resulted in isolation of cDNA species representing two PR-1 homologs. With the exception of one amino acid, the partial amino acid sequences of HvPR-1a and HvPR-1b are identical to internal sequences of the polypeptides derived from the two cDNA species. These derived polypeptides are each 164 amino acids long and both have putative N-terminal leader sequences of 24 amino acids. That these proposed leader sequences are functional is indicated by the observed occurrence of both proteins in the intercellular fluid. The proposed mature proteins (calculated M(r) 14,490 and 15,204) share 91% identical amino acids with each other and 56 to 74% with other PR-1 proteins. Northern blot hybridization and immunoblotting, respectively, show that both transcripts and both proteins accumulate following inoculation of susceptible and hypersensitivity resistant barley leaves with the powdery mildew fungus.

Amino Acid Sequence↗
PubMedDate not supplied

Virus-induced gene expression for enzymes of ethylene biosynthesis in hypersensitively reacting tobacco.

A full-length cDNA clone (cEFE-26) encoding ethylene-forming enzyme (EFE) was isolated from a cDNA library, prepared from leaves of tobacco mosaic virus (TMV)-infected tobacco cultivar Samsun NN. The cDNA clone encodes a protein with 90% amino acid sequence similarity to established EFEs of tomato and other plants. By using cEFE-26 cDNA and the insert from cDNA clone pACC13 (B. A. Bailey, A. Avni, N. Li, A. K. Mattoo, and J. D. Anderson, Plant Physiol. 100:1615-1616, 1992) encoding tobacco 1-aminocyclopropane-1-carboxylic acid synthase as probes, it was established that tobacco contains small gene families for these proteins. Furthermore, RNA blot analyses indicated that transcript levels in leaves for the two ethylene pathway genes were elevated after infection with TMV. The results are discussed in relation to a possible signalling role of ethylene in induced resistance and gene expression for pathogenesis-related proteins.

Amino Acid Sequence↗
PubMed1999

cDNA cloning of a sorghum pathogenesis-related protein (PR-10) and differential expression of defense-related genes following inoculation with Cochliobolus heterostrophus or Colletotrichum sublineolum.

A sorghum cDNA clone was isolated by differential screening of a cDNA library prepared from mesocotyls (cultivar DK18) inoculated with fungal pathogenes. The deduced translation product shows sequence similarity to a family of intracellular pathogenesis-related proteins (PR-10) with a potential ribonuclease function. We studied the accumulation of PR-10 and chalcone synthase (CHS) transcripts in mesocotyls following inoculation with Cochliobolus heterostrophus or Colletotrichum sublineolum. CHS is involved in phytoalexin synthesis in sorghum. Coordinate expression of PR-10 and CHS genes was localized in the area of inoculation along with the accumulation of phytoalexins. C. heterostrophus is a nonpathogen of sorghum and cytological studies indicated that cultivar DK18 is resistant to C. sublineolum, a sorghum pathogen. We demonstrated that the two fungi triggered different time courses of plant defense reactions. Inoculation with C. heterostrophus resulted in rapid accumulation of PR-10 and CHS transcripts after appressoria had become mature. Accumulation of these transcripts was delayed in plants inoculated with C. sublineolum until penetration of host tissue had been completed and infection vesicles had formed. Results suggest that different recognition events are involved in the expression of resistance to the two fungi used or that C. sublineolum suppresses the nonspecific induction of defense responses.

Amino Acid Sequence↗
PubMed2002

A novel short-chain alcohol dehydrogenase from rats with retinol dehydrogenase activity, cyclically expressed in uterine epithelium.

Retinoic acid is necessary for the maintenance of many lining epithelia of the body, such as the epithelium of the luminal surface of the uterus. Administration of estrogen to prepubertal rats induces in these epithelial cells the ability to synthesize retinoic acid from retinol, coincident with the appearance of cellular retinoic acid-binding protein, type two, which is normally present in these cells only at estrus in the mature, cycling animal. Here, we report the isolation, from a cDNA library prepared from uterine mRNA collected at the estrous stage and from a rat mammary adenocarcinoma cell line, of a cDNA that encodes a novel retinol dehydrogenase. A member of the short-chain alcohol dehydrogenase family, the encoded enzyme was capable of metabolizing retinol to retinal when expressed in cells after transfection of its cDNA. When cotransfected with the cDNA of human aldehyde 6, a known retinaldehyde dehydrogenase, the transfected cells synthesized retinoic acid from retinol. Immunohistochemical analysis revealed that the protein was present in the uterine lining epithelium of the mature animal only at estrus, coincident with the presence of cellular retinol-binding protein and cellular retinoic acid-binding protein, type two. Consequently, this novel short-chain alcohol dehydrogenase is an excellent candidate for the retinol dehydrogenase that catalyzes the first step in retinoic acid biosynthesis that occurs in uterine epithelial cells.

Adenocarcinoma↗
PubMed1994

Purification and molecular cloning of bovine oviduct-specific glycoprotein.

A specific 85-97-kDa (95-kDa) glycoprotein was found in bovine oviductal tissue and fluid during the follicular phase. In this study, a 95-kDa bovine oviductal glycoprotein (95-kDa BOGP) was purified by wheat germ agglutinin affinity and Mono-Q ion-exchange column chromatography. The first 29 NH2-terminal amino acid residues were determined by gas-phase microsequencing. A cDNA expression library prepared from poly(A)+ RNA isolated from bovine oviducts was screened with a monoclonal antibody to 95-kDa BOGP. A single positive clone containing a approximately 2-kb cDNA insert was isolated. The coding region contained 1612 bp translating to 537 amino acids. The derived amino acid sequence contained a partial signal sequence of 18 amino acids followed by 29 amino acids that were identical to the NH2-terminal amino acids determined by protein sequencing of purified 95-kDa BOGP. The amino acid sequence predicted a mature protein of 519 amino acids (57,684 daltons) containing one potential N-linked glycosylation site and five cysteines. Northern blot hybridization with a digoxigenin-labeled probe indicated that a single message of approximately 2.5 kb was present in oviductal RNA, and this message was detected in significantly greater amounts in oviductal RNA during the follicular phase than during the luteal phase. The amino acid sequence of a portion of 95-kDa BOGP was highly homologous (71% identity) to that of a baboon oviduct-specific glycoprotein.

Amino Acid Sequence↗
PubMed2000

Cloning and characterization of a complementary deoxyribonucleic acid encoding haploid-specific alanine-rich acidic protein located on chromosome-X.

We have isolated a cDNA clone encoding a germ cell-specific protein from an expression cDNA library prepared from the mouse testis using testis-specific polyclonal antibodies. Northern blot analysis showed a transcript of 1.1 kilobases exclusively expressed in haploid germ cells of the testis. Sequence analysis of the cDNA revealed one long open reading frame consisting of 238 deduced amino acids, rich in basic amino acids in the N-terminal one-third that also contained the nuclear localization signal, and rich in acidic amino acids, including two type of acidic alanine-rich repeats, in the rest of the deduced protein. The protein having a molecular weight of approximately 55 kDa and an isoelectric point of pH 4.3-4.7 was also exclusively detected in the testis by Western blot analysis. As the cDNA was located on chromosome-X, Halap-X (haploid-specific alanine-rich acidic protein located on chromosome-X) was proposed for the name of the protein encoded by the cDNA. Immunohistochemical observation revealed that the Halap-X protein was predominantly present in the nucleoplasm of round spermatids but gradually decreased as spermatids matured, followed by the subsequent appearance in the cytoplasm of elongating spermatids. Thus, the Halap-X protein was transferred from the nuclei to the cytoplasm during the spermatid maturation when the chromatin condensation and transformation of the nuclei occurred. The Halap-X may facilitate specific association of nuclear DNA with some basic chromosomal proteins and play important roles in the process of chromatin condensation.

Amino Acid Sequence↗
PubMed1995

Protection against HIV-1 infection in hu-PBL-SCID mice by passive immunization with a neutralizing human monoclonal antibody against the gp120 CD4-binding site.

OBJECTIVE: Mice with severe combined immunodeficiency (SCID) transplanted with human peripheral blood lymphocytes (hu-PBL) have been shown to be useful as an animal model for HIV-1 infection. This model was used to assess the ability of a human anti-gp120 antibody to protect against HIV-1 infection. DESIGN AND METHODS: hu-PBL-SCID mice were injected with an HIV-1 broadly neutralizing human monoclonal antibody against the gp120 CD4-binding site prior to challenge with HIV-1SF2. The antibody b12, employed for these studies, was isolated from an antibody phage-display library prepared from bone-marrow of a long-term asymptomatic HIV-1-seropositive donor. Both Fab fragments and whole immunoglobulin (Ig) G1 b12 antibody were assessed for protection. RESULTS: Fab b12, tested at a dose approximately 1.9 mg/kg, was able to protect 25% of hu-PBL-SCID mice from HIV-1 infection. IgG1 b12, which displayed favorable pharmacokinetic properties, showed a dose-dependent protection that was complete with a regimen of two injections of 100 micrograms per mouse. The in vivo protective dose of antibody at the time of virus challenge was estimated to be 4.5-7 mg/kg from antibody clearance data. CONCLUSIONS: This study demonstrates for the first time that complete protection against HIV-1 infection can be achieved in the hu-PBL-SCID model by passive immunization with physiologically relevant doses of a human gp120 CD4-binding site antibody derived from natural infection.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Expression of elongation factor-1 gamma-related sequence in human pancreatic cancer.

A cDNA clone designated pPDC-1 was isolated from a cDNA library prepared against poly(A+)RNA isolated from the human pancreatic adenocarcinoma cell line, Capan-2. The cDNA corresponds to a 1.7-kilobase mRNA that is expressed at higher levels in seven of nine pancreatic tumors than in their corresponding normal tissues. It is also expressed in normal human kidney, intestine, pancreas, stomach, placenta, lung, brain, spleen, and liver. A computer search of the Intelligenetics System of all available nucleotide sequences revealed a 60% homology between the nucleotide sequence of the pPDC-1 cDNA and that of elongation factor-1 gamma from Artemia. The deduced amino acid sequence shared 53% identity with the amino acid sequence for the Artemia elongation factor-1 gamma.

Aged↗