Matthew R. Walker

Cloning of cDNA coding for an allergen of cocksfoot grass (Dactylis glomerata) pollen

Messenger RNA isolated from Cocksfoot grass (Dactylis glomerata) anthers has been used to generate a cDNA library in lambda t11. Three cDNA clones (7.8, 8.1, and 8.3) were demonstrated to be recognized by human IgE antibodies in atopic serum and by rabbit polyclonal antiserum raised to a crude aqueous extract of Cocksfoot pollen. The size of the cDNA inserts was determined as approximately 700 bp, and restriction mapping demonstrated them to be identical sequences. Lysogens obtained in Escherichia coli Y1089 allowed expression of a 140 kD beta-galactosidase fusion protein containing 24 kD of cloned allergen protein. Fusion proteins were recognized by IgE antibodies in 75% (6/8) of atopic sera tested, but were not detected by nonatopic sera. On the basis of size and frequency of recognition in the atopic population, the cloned protein may present a major allergen. Monoclonal antibodies specific for the major allergen of Cocksfoot pollen were not reactive with the fusion proteins. Reactivity of human IgE antibodies with the fusion protein could be blocked by crude Cocksfoot pollen extract, but not by the major allergen DG3 purified from the extract by affinity chromatography. Human and rabbit antibodies affinity purified against fusion protein 7.8 did not allow identification of the native protein component in crude extract encoded for by the cDNA clones.

Monoclonal Antibodies to Proteins from Cocksfoot Grass (Dactylis glomerata) Pollen: Isolation and N-Terminal Sequence of a Major Allergen

Monoclonal antibodies (Mabs) raised to an aqueous extract of cocksfoot grass (Dactylis glomerata) pollen have been characterised. Mab 1B9 was demonstrated by SDS-PAGE and Western blotting to recognise a major allergen of an approximate molecular weight of 28 kD in this extract, termed DG3, and a component with a molecular weight of between 35 and 40 kD in an extract of Secale cereale (cultivated rye) pollen. The 28 kD component of cocksfoot grass pollen isolated by affinity chromatography using Mab 1B9 was recognised by IgE antibodies in 80% (8 of 10) atopic sera, but only weakly by 25% (1 of 4) non-atopic sera tested. N-terminal sequencing of DG3 purified by affinity chromatography, 2 D electrophoresis and electroblotting to polyvinylidenedifluoride revealed significant homology with a group-V allergen (Phl p V) from timothy grass (Phleum pratense).

Route maps in gene technology

Preface About the Route Maps format The Concept Of Genes Is DevelopedGenes Are Located To ChromosomesGenes Are Composed Of DNAThe Chemical Building Blocks Of Nucelic AcidsFormation Of The DNA Double HelixPackaging Of DNA Within CellsChromatin Structure And The Functional Activity Of GenesTypes And Functions Of DNA-Protein InteractionsOrganization Of Genomes Into Multiple ChromosomesDistribution Of Nucleic Acids Within Eukaryotic CellsTypes Of RNA MoleculesThe Anatomy Of Eukaryotic ChromosomesThe Organisation Of Genes Within ChromosomesThe Molecular Anatomy Of Eukaryotic GenesChromosome Aberrations And Human DiseaseTypes Of Mutations And Their EffectsForms Of Chemically Altered DNA DNA Repair MechanismsLinkage AnalysisPedigree Analysis And Modes Of InheritanceGenes Dictate The Nature Of ProteinsThe Nature Of The Genetic CodeTranscription: Forming Genetic MessagesPost-Transcriptional Processing Of Messenger RNATransfer And Ribosomal RNA Processing/ModificationMechanisms Regulating Gene ExpressionTranscriptional Regulatory SequencesOperons And Prokaryotic Control Of Gene ExpressionTranscription Factors And Gene ExpressionIn Vivo Translation: Decoding Genetic MessagesSequences Involved In Cellular Protein TargetingEukaryotic Cell Division: Mitosis And MeiosisMolecular Mechanisms Of Cell Cycle ControlGenetic Recombination MechanismsGene Transfer During Bacterial ReproductionTransposable Genetic Elements: TransposonsIn Vivo DNA ReplicationGenetic Control Of DevelopmentThe Natural Biology Of BacteriophagesBacteriophage GeneticsRecombinant DNA TechnologyEnzymes Commonly Used In Molecular Biology MethodsRestriction EndonucleasesRestriction Fragment Length PolymorphismsIsolation Of Nucleic Acids From Cells And TissuesVisualising Mucleic AcidsElectrophoresis Of Nucleic AcidsIn Vitro HybridisationTypes Of Hybridisation Assay FormatsSouthern Blotting In Situ HybridisationMeasuring Transcriptional Activity Via Messenger RNAConverting Messenger RNA Into Complementary DNA (Cdna)Methods For Determining DNA Nucleotide SequencesThe Polymerase Chain ReactionAlternatives To PCR-Based In Vitro DNA/RNA AmplificationIn Vitro Translation MethodsTypes And Methods Of Gene Probe GenerationChemical Synthesis Of OligonucleotidesTypes And Applications Of Nucleotide AnaloguesMethods For Labelling Gene ProbesFundamental Principles Of CloningThe Nature Of Cloning VectorsInserting Foreign DNA Into VectorsThe Development Of Bacteriophage VectorsPlasmids: Development As Clonign VectorsYeast-Derived Plasmid VectorsPhagemids: Hybrid Phage And Plasmid VectorsVectors For Use In Plant And Animal CellsDelivering DNA Into Cells Principal Genomic And Cdna Cloning StrategiesStrategies For Identifying Desirable Recombinant ClonesGene Mapping TechniquesDetecting DNA-Protein Interaction SitesDetecting Promoter And Enhancer SequencesMethods For Identifying Protein Encoding SequencesGenetic FingerprintingAnalysing Ancient DnasIn Vitro Mutagenesis MethodsGenetically Modified Micro-OrganismsGenetically Engineered PlantsGenetically Engineered AnimalsMolecular Techniques In Prenatal DiagnosisThe Genetics Of Cystic FibrosisThe Dystrophin Gene And Muscular DystrophiesIdentifying The Gene For Huntingdons DiseaseLipoprotein Genes And Heart DiseaseThe Detection Of Microbial InfectionsMolecular Biology Of Human Immunodeficiency Virus And AIDSEngineering Microbial BioluminescenceRecombinant DNA Techniques In Vaccine DevelopmentReceptors And Cellular Signalling MechanismsOncogenes And The Molecular Basis Of CancerMolecular Diagnosis And Therapy Of CancersDrug Development Using Recombinant DNA ApproachesProtein EngineeringImmunoglobulin GeneticsGenetic Engineering Of Recombinant AntibodiesCurrent Approaches To Gene TherapyThe Human Genome Mapping ProjectBibliography Index

Isolation and amplification of human IgE Fd encoding mRNA from human peripheral blood lymphocytes

In order to establish the feasibility of applying recombinatorial library technologies to investigate human in vivo IgE responses, and as a pre-requisite of recombinatorial library construction, we have attempted to determine workable peripheral blood sample volumes required for isolation of mRNA for polymerase chain reaction (PCR) amplification of human IgE Fd encoding sequences. Cells secreting chimeric human IgE monoclonal antibody specific for the hapten NIP were used to establish the conditions for specific amplification of C epsilon 1 domain and Fd encoding sequences, as determined by Southern hybridisation. Amplification of C epsilon 1 domain sequences could be achieved using as few as ten cultured cells as the source of RNA. Specific IgE+ B cell enrichment using immuno-magnetic particles prior to RNA extraction was, however, required to obtain amplification of IgE C epsilon 1 and Fd fragments from lymphocytes prepared from 40 ml human peripheral blood. IgG1+ B cell enrichment from similar samples was not required for detectable amplification of human C gamma 1 cDNA sequences. However, this procedure improved amplification efficiency. Optimisation of methods to separate specific B cell populations, or specific RNA/cDNA sequences, will facilitate in vitro generation of human IgE Fab fragments from peripheral blood.