Biomaterial Database

Expression analysis of a pseudogene in transgenic tobacco: a frameshift mutation prevents mRNA accumulation. 1990

T A Voelker, and J Moreno, and M J Chrispeels

Department of Biology, University of California, San Diego, La Jolla 92093-0116.

Seeds of the Pinto cultivar of the common bean, Phaseolus vulgaris, are deficient in phytohemagglutinin (PHA), a lectin normally composed of two different polypeptides (PHA-E and PHA-L). In Pinto seeds, there is no PHA-E and only small amounts of PHA-L. The gene coding for the Pinto PHA-E, Pdlec1, is a pseudogene as a result of a single base pair deletion in codon 11, causing a frameshift and premature termination of translation. This mutation explains the absence of the PHA-E polypeptide but not the several-hundredfold reduction of the cytoplasmic Pdlec1 mRNA in developing seeds when compared with a normal PHA-E gene. To find the cause for this reduction in mRNA levels, we swapped gene fragments of Pdlec1 with the homologous parts of a normal PHA gene from the cultivar Greensleeves and introduced these fusions into tobacco. Analysis of the transgenic seeds showed that the Pdlec1 promoter is fully functional. We also repaired the Pdlec1 coding frame in vitro and inserted the repaired and unrepaired versions into a PHA gene expression cassette. In transgenic tobacco, both constructs showed Pdlec1 transcript accumulation in the second half of seed maturation. The single-base frame repair boosted the peak transcript levels by a factor of 40 and resulted in the synthesis of PHA-E at normal levels. We propose that the premature translational stop caused by the frameshift leads to a faster breakdown of the Pdlec1 mRNA, thereby preventing this transcript from accumulating to high levels.

UI	MeSH Term	Description	Entries
D010835	Phytohemagglutinins	Mucoproteins isolated from the kidney bean (Phaseolus vulgaris); some of them are mitogenic to lymphocytes, others agglutinate all or certain types of erythrocytes or lymphocytes. They are used mainly in the study of immune mechanisms and in cell culture.	Kidney Bean Lectin,Kidney Bean Lectins,Lectins, Kidney Bean,Phaseolus vulgaris Lectin,Phaseolus vulgaris Lectins,Phytohemagglutinin,Hemagglutinins, Plant,Lectin, Kidney Bean,Lectin, Phaseolus vulgaris,Lectins, Phaseolus vulgaris,Plant Hemagglutinins
D010947	Plants, Toxic	Plants or plant parts which are harmful to man or other animals.	Plants, Poisonous,Plant, Poisonous,Plant, Toxic,Poisonous Plant,Poisonous Plants,Toxic Plant,Toxic Plants
D011544	Pseudogenes	Genes bearing close resemblance to known genes at different loci, but rendered non-functional by additions or deletions in structure that prevent normal transcription or translation. When lacking introns and containing a poly-A segment near the downstream end (as a result of reverse copying from processed nuclear RNA into double-stranded DNA), they are called processed genes.	Genes, Processed,beta-Tubulin Pseudogene,Gene, Processed,Processed Gene,Processed Genes,Pseudogene,Pseudogene, beta-Tubulin,Pseudogenes, beta-Tubulin,beta Tubulin Pseudogene,beta-Tubulin Pseudogenes
D011993	Recombinant Fusion Proteins	Recombinant proteins produced by the GENETIC TRANSLATION of fused genes formed by the combination of NUCLEIC ACID REGULATORY SEQUENCES of one or more genes with the protein coding sequences of one or more genes.	Fusion Proteins, Recombinant,Recombinant Chimeric Protein,Recombinant Fusion Protein,Recombinant Hybrid Protein,Chimeric Proteins, Recombinant,Hybrid Proteins, Recombinant,Recombinant Chimeric Proteins,Recombinant Hybrid Proteins,Chimeric Protein, Recombinant,Fusion Protein, Recombinant,Hybrid Protein, Recombinant,Protein, Recombinant Chimeric,Protein, Recombinant Fusion,Protein, Recombinant Hybrid,Proteins, Recombinant Chimeric,Proteins, Recombinant Fusion,Proteins, Recombinant Hybrid
D003001	Cloning, Molecular	The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.	Molecular Cloning
D012333	RNA, Messenger	RNA sequences that serve as templates for protein synthesis. Bacterial mRNAs are generally primary transcripts in that they do not require post-transcriptional processing. Eukaryotic mRNA is synthesized in the nucleus and must be exported to the cytoplasm for translation. Most eukaryotic mRNAs have a sequence of polyadenylic acid at the 3' end, referred to as the poly(A) tail. The function of this tail is not known for certain, but it may play a role in the export of mature mRNA from the nucleus as well as in helping stabilize some mRNA molecules by retarding their degradation in the cytoplasm.	Messenger RNA,Messenger RNA, Polyadenylated,Poly(A) Tail,Poly(A)+ RNA,Poly(A)+ mRNA,RNA, Messenger, Polyadenylated,RNA, Polyadenylated,mRNA,mRNA, Non-Polyadenylated,mRNA, Polyadenylated,Non-Polyadenylated mRNA,Poly(A) RNA,Polyadenylated mRNA,Non Polyadenylated mRNA,Polyadenylated Messenger RNA,Polyadenylated RNA,RNA, Polyadenylated Messenger,mRNA, Non Polyadenylated
D012639	Seeds	The encapsulated embryos of flowering plants. They are used as is or for animal feed because of the high content of concentrated nutrients like starches, proteins, and fats. Rapeseed, cottonseed, and sunflower seed are also produced for the oils (fats) they yield.	Diaspores,Elaiosomes,Embryos, Plant,Plant Embryos,Plant Zygotes,Zygotes, Plant,Diaspore,Elaiosome,Embryo, Plant,Plant Embryo,Plant Zygote,Seed,Zygote, Plant
D014026	Nicotiana	A plant genus of the family SOLANACEAE. Members contain NICOTINE and other biologically active chemicals; the dried leaves of Nicotiana tabacum are used for SMOKING.	Tobacco Plant,Nicotiana tabacum,Plant, Tobacco,Plants, Tobacco,Tobacco Plants
D015151	Immunoblotting	Immunologic method used for detecting or quantifying immunoreactive substances. The substance is identified by first immobilizing it by blotting onto a membrane and then tagging it with labeled antibodies.	Dot Immunoblotting,Electroimmunoblotting,Immunoelectroblotting,Reverse Immunoblotting,Immunoblotting, Dot,Immunoblotting, Reverse,Dot Immunoblottings,Electroimmunoblottings,Immunoblottings,Immunoblottings, Dot,Immunoblottings, Reverse,Immunoelectroblottings,Reverse Immunoblottings
D015183	Restriction Mapping	Use of restriction endonucleases to analyze and generate a physical map of genomes, genes, or other segments of DNA.	Endonuclease Mapping, Restriction,Enzyme Mapping, Restriction,Site Mapping, Restriction,Analysis, Restriction Enzyme,Enzyme Analysis, Restriction,Restriction Enzyme Analysis,Analyses, Restriction Enzyme,Endonuclease Mappings, Restriction,Enzyme Analyses, Restriction,Enzyme Mappings, Restriction,Mapping, Restriction,Mapping, Restriction Endonuclease,Mapping, Restriction Enzyme,Mapping, Restriction Site,Mappings, Restriction,Mappings, Restriction Endonuclease,Mappings, Restriction Enzyme,Mappings, Restriction Site,Restriction Endonuclease Mapping,Restriction Endonuclease Mappings,Restriction Enzyme Analyses,Restriction Enzyme Mapping,Restriction Enzyme Mappings,Restriction Mappings,Restriction Site Mapping,Restriction Site Mappings,Site Mappings, Restriction