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PubMed2001

Molecular definition of the germinal centre stage of B-cell differentiation.

Genomic-scale gene expression analysis provides views of biological processes as a whole that are difficult to obtain using traditional single-gene experimental approaches. In the case of differentiating systems, gene expression profiting can define a stage of differentiation by the characteristic expression of hundreds of genes. Using specialized DNA microarrays termed 'Lymphochips', gene expression during mature B-cell differentiation has been defined. Germinal centre B cells represent a stage of differentiation that can be defined by a gene expression signature that is not shared by other highly proliferative B-cell populations such as mitogenically activated peripheral blood B cells. The germinal centre gene expression signature is maintained to a significant degree in lymphoma cell lines derived from this stage of differentiation, demonstrating that this gene expression programme does not require ongoing interactions with other germinal centre cell types. Analysis of representative cDNA libraries prepared from resting and activated peripheral blood B cells, germinal centre centroblasts, centrocytes and tonsillar memory B cells has confirmed and extended the results of DNA microarray gene expression analysis.

B-Lymphocytes↗
PubMed2002

From genomes to vaccines: Leishmania as a model.

The 35 Mb genome of Leishmania should be sequenced by late 2002. It contains approximately 8500 genes that will probably translate into more than 10 000 proteins. In the laboratory we have been piloting strategies to try to harness the power of the genome-proteome for rapid screening of new vaccine candidate. To this end, microarray analysis of 1094 unique genes identified using an EST analysis of 2091 cDNA clones from spliced leader libraries prepared from different developmental stages of Leishmania has been employed. The plan was to identify amastigote-expressed genes that could be used in high-throughput DNA-vaccine screens to identify potential new vaccine candidates. Despite the lack of transcriptional regulation that polycistronic transcription in Leishmania dictates, the data provide evidence for a high level of post-transcriptional regulation of RNA abundance during the developmental cycle of promastigotes in culture and in lesion-derived amastigotes of Leishmania major. This has provided 147 candidates from the 1094 unique genes that are specifically upregulated in amastigotes and are being used in vaccine studies. Using DNA vaccination, it was demonstrated that pooling strategies can work to identify protective vaccines, but it was found that some potentially protective antigens are masked by other disease-exacerbatory antigens in the pool. A total of 100 new vaccine candidates are currently being tested separately and in pools to extend this analysis, and to facilitate retrospective bioinformatic analysis to develop predictive algorithms for sequences that constitute potentially protective antigens. We are also working with other members of the Leishmania Genome Network to determine whether RNA expression determined by microarray analyses parallels expression at the protein level. We believe we are making good progress in developing strategies that will allow rapid translation of the sequence of Leishmania into potential interventions for disease control in humans.

Animals↗
PubMed1992

Mapping of sequential epitopes recognized by monoclonal antibodies on the bovine leukaemia virus external glycoproteins expressed in Escherichia coli by means of antipeptide antibodies.

A lambda gt11 cDNA library prepared from bovine leukaemia virus (BLV)-producing ovine cells was screened with a cocktail of anti-BLV gp51 monoclonal antibodies (MAbs). Four recombinant phages with inserts of about 2-5 kbp were isolated. One, lambda BLV-gp51-1, was sequenced and shown to encode the C-terminal part of gp51 and all of gp30. This insert was subcloned into pEV-vrf1 and expressed in Escherichia coli N-4830-1 cells. The BLV product and a series of antipeptide antibodies were used to localize the sequential epitopes defined on BLV envelope glycoprotein gp51 by their reactivity with MAbs. Epitope B was localized to amino acids 180 to 205, B' to residues 195 to 205, D and D' to residues 218 to 237, and A to amino acids 249 to 260. All the mapped sequential epitopes were localized in the C-terminal half of BLV gp51. The results of epitope mapping with bacterially produced gp51 confirm the map obtained using native viral glycoprotein.

Amino Acid Sequence↗
PubMed1998

Human recombinant Puumala virus antibodies: cross-reaction with other hantaviruses and use in diagnostics.

A panel of seven human monoclonal Fabs against Puumala virus (PUU) nucleocapsid protein (N) was obtained by panning an antibody phage-display library prepared from the spleen of a PUU-immune individual. Three antibodies reacted in immunoblotting and cross-reacted strongly with Tula and Sin Nombre virus recombinant N proteins. These antibodies mapped to the amino terminus of the N protein. One PUU glycoprotein 2 (G2)-specific Fab obtained against a novel epitope (G2c) cross-reacted with Khabarovsk virus but not with the other hantavirus serotypes. An N protein-specific Fab was successfully used as capture antibody to detect PUU-specific serum IgG and IgM antibodies in an enzyme immunoassay. The result demonstrates the usefulness of recombinant human Fabs as potential diagnostic tools.

Antibodies, Monoclonal↗
PubMed1996

Transcriptional activation of the Cdk inhibitor p21 by vitamin D3 leads to the induced differentiation of the myelomonocytic cell line U937.

The hormonal form of vitamin D, 1,25-dihydroxyvitamin D3, acting through its cognate nuclear receptor (vitamin D3 receptor, VDR) will induce myeloid leukemic cell lines to terminally differentiate into monocytes/macrophages. Because VDR acts by transcriptionally regulating responsive genes in a ligand-dependent manner, we sought target genes of the receptor that initiate, the differentiation process in response to ligand. We screened a cDNA library prepared from the myelomonocytic U937 cell line with probes generated from either 1,25-dihydroxyvitamin D3-treated or untreated cells. We report here that a candidate clone that hybridized differentially is the Cdk inhibitor p21WAF1, CIP1. Furthermore, we show that p21 is transcriptionally induced by 1,25-dihydroxyvitamin D3 in a VDR-dependent, but not p53-dependent, manner, and we identify a functional vitamin D response element in the p21 promoter. Transient overexpression of p21 and/or the related Cdk inhibitor p27 in U937 cells in the absence of 1,25-dihydroxyvitamin D3 results in the cell-surface expression of monocyte/macrophage-specific markers, suggesting that ligand-modulated transcriptional induction of the p21 gene facilitates the induced differentiation of this monoblastic cell line. We believe that this is the first report demonstrating that the ectopic overexpression of a Cdk inhibitor such as p21 or p27 directly leads to a terminal differentiation program.

Base Sequence↗
PubMed2005

The developmental miRNA profiles of zebrafish as determined by small RNA cloning.

MicroRNAs (miRNAs) represent a family of small, regulatory, noncoding RNAs that are found in plants and animals. Here, we describe the miRNA profile of the zebrafish Danio rerio resolved in a developmental and cell-type-specific manner. The profiles were obtained from larger-scale sequencing of small RNA libraries prepared from developmentally staged zebrafish, and two adult fibroblast cell lines derived from the caudal fin (ZFL) and the liver epithelium (SJD). We identified a total of 154 distinct miRNAs expressed from 343 miRNA genes. Other experimental/computational sources support an additional 10 miRNAs encoded by 19 genes. The miRNAs can be classified into 87 distinct families. Cross-species comparison indicates that 81 families are conserved in mammals, 17 of which also have at least one member conserved in an invertebrate. Our analysis reveals that the zygotes are essentially devoid of miRNAs and that their expression begins during the blastula period with a zebrafish-specific family of miRNAs encoded by closely spaced multicopy genes. Computational predictions of zebrafish miRNA targets are provided that take into account the depth of evolutionary conservation. Besides miRNAs, we identified a prominent class of repeat-associated small interfering RNAs (rasiRNAs).

Animals↗
PubMed1989

A gene with homology to the myc similarity region of MyoD1 is expressed during myogenesis and is sufficient to activate the muscle differentiation program.

MyoD1 is a nuclear phosphoprotein that is expressed in skeletal muscle in vivo and in certain muscle cell lines in vitro; it has been shown to convert fibroblasts to myoblasts through a mechanism requiring a domain with homology to the myc family of proteins. The BC3H1 muscle cell line expresses skeletal muscle-specific genes upon exposure to mitogen-deficient medium, but does not express MyoD1 at detectable levels. To determine whether BC3H1 cells may express regulatory genes functionally related to MyoD1, a cDNA library prepared from differentiated BC3H1 myocytes, was screened at reduced stringency with the region of the MyoD1 cDNA that shares homology with c-myc. From this screen, a cDNA was identified that encodes a major open reading frame with 72% homology to the myc domain and basic region of MyoD1. The mRNA encoded by this MyoD1-related gene is expressed in skeletal muscle in vivo and in differentiated skeletal myocytes in vitro and is undetectable in cardiac or smooth muscle, nonmuscle tissues, or nonmyogenic cell types. During myogenesis, the MyoD1-related mRNA accumulates several hours prior to other muscle-specific mRNAs and therefore represents an early molecular marker for entry of myoblasts into the differentiation pathway. Transient transfection of 10T1/2 or 3T3 cells with the MyoD1-related cDNA is sufficient to induce myosin heavy-chain expression and to activate a reporter gene under transcriptional control of the muscle creatine kinase 5' enhancer, which functions only in differentiated myocytes. Expression of this cDNA in stably transfected 10T1/2 cells also leads to fusion and muscle-specific gene expression upon exposure to mitogen-deficient medium. Thus, the product of this MyoD1-related gene is sufficient to activate the muscle differentiation program and may substitute for MyoD1 in certain developmental situations. Together, these results suggest the existence of a family of myogenic regulatory genes that share a conserved motif with c-myc.

Amino Acid Sequence↗
PubMed1994

Peach (Prunus persica) endopolygalacturonase cDNA isolation and mRNA analysis in melting and nonmelting peach cultivars.

Two distinct partial cDNAs, PRF1 and PRF3, similar in sequence to previously described polygalacturonases, were amplified from ripe peach (Prunus persica L. Batsch cv Flavorcrest) fruit cDNA by the polymerase chain reaction. PRF1-related RNA was present in fruit from early ripening at levels not detected by northern analysis. PRF3-related RNA was readily detectable in ripe fruit by northern analysis. PRF3 was used to isolate a cDNA with a complete open reading frame, PRF5, from a lambda ZAP II cDNA library prepared from poly(A)+ RNA of ripe peach fruit. PRF5 coded for a predicted protein of 393 amino acids with a molecular mass of 41,500 D. The derived amino acid sequence of PRF5 included a putative leader sequence of 23 amino acids, followed by a sequence that matched the N terminus of endopolygalacturonase protein purified from ripe peach fruit. By northern analysis, PRF3-related RNA was undetectable in firm, unripe Flavorcrest fruit. It appeared at low levels as a 1.7-kb transcript in fruit that had begun to ripen and soften and was very abundant in ripe fruit that had undergone the "melting" stage of softening. The marked increase in PRF3-related RNA levels took place over a period of less than 2 d at 20 degrees C and coincided with the climacteric peak in ethylene evolution. Levels of 1-aminocyclopropane-1-carboxylate oxidase-related RNA increased during ripening at a much earlier stage than levels of PRF3-related RNA. Lower levels of 1.7-kb RNA transcript were detected by PRF3 in ripe fruit of the melting cultivar Fragar, which are firmer than Flavorcrest fruit. In ripe fruit of the nonmelting cultivar Carolyn, PRF3 detected a 1.45-kb RNA transcript that was present at low levels. Transcripts of a peach polygalacturonase-related genomic sequence were not detected in ripening fruit.

Amino Acid Sequence↗
PubMed1994

Genes galore: a summary of methods for accessing results from large-scale partial sequencing of anonymous Arabidopsis cDNA clones.

High-throughput automated partial sequencing of anonymous cDNA clones provides a method to survey the repertoire of expressed genes from an organism. Comparison of the coding capacity of these expressed sequence tags (ESTs) with the sequences in the public data bases results in assignment of putative function to a significant proportion of the ESTs. Thus, the more than 13,400 plant ESTs that are currently available provide a new resource that will facilitate progress in many areas of plant biology. These opportunities are illustrated by a description of the results obtained from analysis of 1500 Arabidopsis ESTs from a cDNA library prepared from equal portions of poly(A+) mRNA from etiolated seedlings, roots, leaves, and flowering inflorescences. More than 900 different sequences were represented, 32% of which showed significant nucleotide or deduced amino acid sequences similarity to previously characterized genes or proteins from a wide range of organisms. At least 165 of the clones had significant deduced amino acid sequence homology to proteins or gene products that have not been previously characterized from higher plants. A summary of methods for accessing the information and materials generated by the Arabidopsis cDNA sequencing project is provided.

Amino Acid Sequence↗
PubMed1997

Characterization of a maize beta-amylase cDNA clone and its expression during seed germination.

A maize (Zea mays L.) cDNA clone (pZMB2) encoding beta-amylase was isolated from a cDNA library prepared from the aleurone RNA of germinating kernels. The cDNA encodes a predicted product of 488 amino acids with significant similarity to known beta-amylases from barley (Hordeum vulgare), rye (Secale cereale), and rice (Oryza sativa). Glycine-rich repeats found in the carboxyl terminus of the endosperm-specific beta-amylase of barley and rye are absent from the maize gene product. The N-terminal sequence of the first 20 amino acids of a beta-amylase peptide derived from purified protein is identical to the 5th through 24th amino acids of the predicted cDNA product, indicating the absence of a conventional signal peptide in the maize protein. Recombinant inbred mapping data indicate that the cDNA clone is single-copy gene that maps to chromosome 7L at position 83 centimorgans. Northern blot analysis and in vitro translation-immunoprecipitation data indicate that the maize beta-amylase is synthesized de novo in the aleurone cells but not in the scutellum during seed germination.

Amino Acid Sequence↗
PubMed1997

The sorghum photoperiod sensitivity gene, Ma3, encodes a phytochrome B.

The Ma3 gene is one of six genes that regulate the photoperiodic sensitivity of flowering in sorghum (Sorghum bicolor [L.] Moench). The ma3R mutation of this gene causes a phenotype that is similar to plants that are known to lack phytochrome B, and ma3 sorghum lacks a 123-KD phytochrome that predominates in light-grown plants and that is present in non-ma3 plants. A population segregating for Ma3 and ma3 was created and used to identify two randomly amplified polymorphic DNA markers linked to Ma3. These two markers were cloned and mapped in a recombinant inbred population as restriction fragment length polymorphisms. cDNA clones of PHYA and PHYC were cloned and sequenced from a cDNA library prepared from green sorghum leaves. Using a genome-walking technique, a 7941-bp partial sequence of PHYB, was determined from genomic DNA from ma3 sorghum. PHYA, PHYB, and PHYC all mapped to the same linkage group. The Ma3-linked markers mapped with PHYB more than 121 centimorgans from PHYA and PHYC. A frameshift mutation resulting in a premature stop codon was found in the PHYB sequence from ma3 sorghum. Therefore, we conclude that the Ma3 locus in sorghum is a PHYB gene that encodes a 123-kD phytochrome.

Amino Acid Sequence↗
PubMed1998

Two pine endo-beta-1,4-glucanases are associated with rapidly growing reproductive structures.

Two cDNA clones encoding endo-beta-1,4-glucanases (EGases) were isolated from a radiata pine (Pinus radiata) cDNA library prepared from immature female strobili. The cDNAs PrCel1 (inus adiata cellulase ) and PrCel2 encode proteins 509 and 515 amino acids in length, respectively, including putative signal peptides. Both proteins contain domains conserved in plant and bacterial EGases. The proteins PRCEL1 and PRCEL2 showed strong similarity to each other (76% amino acid identity), and higher similarity to TPP18 (73 and 67%, respectively), an EGase cloned from tomato (Lycopersicon esculentum) pistils, than to any other reported EGases. Northern-blot analyses indicated that both genes displayed a similar pattern of expression. The only significant difference was in the level of expression. In situ hybridizations were used to demonstrate that, within differentiating pine reproductive structures, PrCel1 expression was greatest in microsporangia in pollen strobili and near the developing ovule in the seed strobili. Expression was also found in vegetative tissues, especially in regions experiencing cell elongation, such as the elongating region of root tips. Both proteins have an ability to degrade carboxymethylcellulose in vitro. Genomic-blot analysis indicated the presence of a family of EGase genes in the radiata pine genome, and that PrCel1 and PrCel2 are transcribed from distinct one-copy genes.

Bacterial Proteins↗
PubMed1999

Characterization of Chlamydomonas reinhardtii zygote-specific cDNAs that encode novel proteins containing ankyrin repeats and WW domains.

Genes that are expressed only in the young zygote are considered to be of great importance in the development of an isogamous green alga, Chlamydomonas reinhardtii. Clones representing the Zys3 gene were isolated from a cDNA library prepared using zygotes at 10 min after fertilization. Sequencing of Zys3 cDNA clones resulted in the isolation of two related molecular species. One of them encoded a protein that contained two kinds of protein-to-protein interaction motifs known as ankyrin repeats and WW domains. The other clone lacked the ankyrin repeats but was otherwise identical. These mRNA species began to accumulate simultaneously in cells beginning 10 min after fertilization, and reached maximum levels at about 4 h, after which time levels decreased markedly. Genomic DNA gel-blot analysis indicated that Zys3 was a single-copy gene. The Zys3 proteins exhibited parallel expression to the Zys3 mRNAs at first, appearing 2 h after mating, and reached maximum levels at more than 6 h, but persisted to at least 1 d. Immunocytochemical analysis revealed their localization in the endoplasmic reticulum, which suggests a role in the morphological changes of the endoplasmic reticulum or in the synthesis and transport of proteins to the Golgi apparatus or related vesicles.

Amino Acid Sequence↗
PubMed2000

A stress-inducible gene for 9-cis-epoxycarotenoid dioxygenase involved in abscisic acid biosynthesis under water stress in drought-tolerant cowpea.

Four cDNA clones named CPRD (cowpea responsive to dehydration) corresponding to genes that are responsive to dehydration were isolated using differential screening of a cDNA library prepared from 10-h dehydrated drought-tolerant cowpea (Vigna unguiculata) plants. One of the cDNA clones has a homology to 9-cis-epoxycarotenoid dioxygenase (named VuNCED1), which is supposed to be involved in abscisic acid (ABA) biosynthesis. The GST (glutathione S-transferase)-fused protein indicates a 9-cis-epoxycarotenoid dioxygenase activity, which catalyzes the cleavage of 9-cis-epoxycarotenoid. The N-terminal region of the VuNCED1 protein directed the fused sGFP (synthetic green-fluorescent protein) into the plastids of the protoplasts, indicating that the N-terminal sequence acts as a transit peptide. Both the accumulation of ABA and expression of VuNCED1 were strongly induced by drought stress in the 8-d-old cowpea plant, whereas drought stress did not trigger the expression of VuABA1 (accession no. AB030295) gene that encodes zeaxanthin epoxidase. These results indicate that the VuNCED1 cDNA encodes a 9-cis-epoxycarotenoid dioxygenase and that its product has a key role in the synthesis of ABA under drought stress.

Abscisic Acid↗
PubMed2000

Production of fatty acid components of meadowfoam oil in somatic soybean embryos.

The seed oil of meadowfoam (Limnanthes alba) and other Limnanthes spp. is enriched in the unusual fatty acid Delta(5)-eicosenoic acid (20:1Delta(5)). This fatty acid has physical and chemical properties that make the seed oil of these plants useful for a number of industrial applications. An expressed sequence tag approach was used to identify cDNAs for enzymes involved in the biosynthesis of 20:1Delta(5)). By random sequencing of a library prepared from developing Limnanthes douglasii seeds, a class of cDNAs was identified that encode a homolog of acyl-coenzyme A (CoA) desaturases found in animals, fungi, and cyanobacteria. Expression of a cDNA for the L. douglasii acyl-CoA desaturase homolog in somatic soybean (Glycine max) embryos behind a strong seed-specific promoter resulted in the accumulation of Delta(5)-hexadecenoic acid to amounts of 2% to 3% (w/w) of the total fatty acids of single embryos. Delta(5)-Octadecenoic acid and 20:1Delta(5) also composed <1% (w/w) each of the total fatty acids of these embryos. In addition, cDNAs were identified from the L. douglasii expressed sequence tags that encode a homolog of fatty acid elongase 1 (FAE1), a beta-ketoacyl-CoA synthase that catalyzes the initial step of very long-chain fatty acid synthesis. Expression of the L. douglassi FAE1 homolog in somatic soybean embryos was accompanied by the accumulation of C(20) and C(22) fatty acids, principally as eicosanoic acid, to amounts of 18% (w/w) of the total fatty acids of single embryos. To partially reconstruct the biosynthetic pathway of 20:1Delta(5) in transgenic plant tissues, cDNAs for the L. douglasii acyl-CoA desaturase and FAE1 were co-expressed in somatic soybean embryos. In the resulting transgenic embryos, 20:1Delta(5) and Delta(5)-docosenoic acid composed up to 12% of the total fatty acids.

Acetyltransferases↗
PubMed1989

Molecular cloning and relationship to freezing tolerance of cold-acclimation-specific genes of alfalfa.

Cold-acclimation-specific (CAS) gene expression has been examined by screening a cDNA library prepared from poly(A)(+) RNA of cold-acclimated seedlings of a freezing-tolerant variety of alfalfa (Medicago falcata cv Anik). Three CAS cDNA clones, pSM784, pSM2201, and pSM2358, representing different sequence species, have been used to investigate the relative abundance and time-course of accumulation of corresponding transcripts. Results obtained show that the expression of these CAS genes is regulated in a coordinated manner most likely at the level of transcription. The expression of genes, as measured by mRNA abundance corresponding to the three CAS cDNA clones, is not stimulated or induced by heat shock, water stress, abscisic acid, or wounding. A positive correlation is observed between the expression of these cloned sequences and the degree of freezing-tolerance in four alfalfa cultivars.

Journal Article↗
PubMed1989

Molecular cloning and characterization of senescence-related genes from carnation flower petals.

The senescence of carnation (Dianthus caryophyllus L.) flower petals is associated with increased production of ethylene which plays an important role in regulating this developmental event. Three senescence-related cDNA clones were isolated from a cDNA library prepared from mRNA isolated from senescing petals. These cDNAs are representative of two classes of mRNAs which increase in abundance in senescing petal tissue. The mRNA for one class is present at low levels during the early stages of development and begins to accumulate in mature petals prior to the increase in ethylene production. The accumulation of this mRNA is reduced, but not eliminated, in petals treated with aminooxyacetic acid, an inhibitor of ethylene biosynthesis, or silver thiosulfate, an ethylene action inhibitor. In contrast, expression of the second class of mRNAs appears to be highly regulated by ethylene. These mRNAs are not detectable prior to the rise in ethylene production and increase in abundance in parallel with the ethylene climacteric. Furthermore, expression of these mRNAs is significantly inhibited by both aminooxyacetic acid and silver thiosulfate. Expression of these mRNAs in vegetative and floral organs was limited to floral tissue, and predominantly to senescing petals.

Journal Article↗
PubMed1990

Molecular Cloning and Ethylene Induction of mRNA Encoding a Phytoalexin Elicitor-Releasing Factor, beta-1,3-Endoglucanase, in Soybean.

Soybean (Glycine max) beta-1,3-endoglucanase (EC 3.2. 1.39) is involved in one of the earliest plant-pathogen interactions that may lead to active disease resistance by releasing elicitor-active carbohydrates from the cell walls of fungal pathogens. Ethylene induced beta-1,3-endoglucanase activity to 2- to 3-fold higher levels in cotyledons of soybean seedlings. A specific polyclonal antiserum raised against purified soybean beta-1,3-endoglucanase was used to immunoprecipitate in vitro translation products, demonstrating that ethylene induction increased translatable beta-1,3-endoglucanase mRNA. Several cDNA clones for the endoglucanase gene were obtained by antibody screening of a lambda-gt11 expression library prepared from soybean cotyledons. Hybrid-select translation experiments indicated that the cloned cDNA encoded a 36-kilodalton precursor protein product that was specifically immunoprecipitated with beta-1,3-endoglucanase antiserum. Escherichia coli cells expressing the cloned cDNA also synthesized an immunologically positive protein. Nucleotide sequence of three independent clones revealed a single uninterrupted open reading frame of 1041 nucleotides, corresponding to a polypeptide of 347 residue long. The primary amino acid sequence of beta-1,3-endoglucanase as deduced from the nucleotide sequence was confirmed by direct amino acid sequencing of trypsin digests of the glucanase. The soybean beta-1,3-endoglucanase exhibited 53% amino acid homology to a beta-1,3-glucanase cloned from cultured tobacco cells and 48% homology to a beta-(1,3-1,4)-glucanase from barley. Utilizing the largest cloned cDNA (pEG488) as a hybridization probe, it was found that the increase in translatable beta-1,3-endoglucanase mRNA seen upon ethylene treatment of soybean seedlings was due to 50- to 100-fold increase in steady state mRNA levels, indicating that ethylene regulates gene expression of this enzyme important in disease resistance at the level of gene transcription.

Journal Article↗