Occurrence and expression of members of the ferritin gene family in cowpeas.

Ferritin gene expression has been demonstrated in a variety of plants including maize, Arabidopsis, cowpeas, soybeans, beans and peas. Most available evidence shows that the mature protein is located in plastids and its production is under gene transcriptional control. In maize, two different ferritin genes have been identified; they were found to express protein under different physiological conditions. Only single gene products have been found until now in the other plants, with the exception of cowpeas (Vigna unguiculata). Our previous work with cowpeas [Wicks and Entsch (1993) Biochem. Biophys. Res. Commun. 192, 813-819] showed the existence of a family of at least three ferritin genes, each coding for a protein subunit with a unique amino acid sequence. Here we report the discovery of a fourth active gene in cowpeas and present the full cDNA sequences for two of the four known members of the cowpea gene family. We also provide preliminary evidence for a family of ferritin genes in soybeans (Glycine max) related to that in cowpeas. We conclude that a family of genes is probably present in all higher plants. We have used quantitative reverse transcriptase-mediated PCR to show that each of the four members of the cowpea ferritin gene family expresses mRNA in leaves and roots under normal growth with a complete nutrient supply. The results clearly show a marked differential pattern of mRNA levels formed during development from the four genes. We conclude that the composition of plant ferritin molecules from plant leaf extracts is probably a complex mixture of subunits, which might be different in roots and in leaves.

A hybridisation technique to identify anthelmintic resistance genes in Haemonchus.

The identification of genes associated with anthelmintic resistance can be facilitated in Haemonchus contortus by the ability of this species to hybridise with Haemonchus placei. Although the hybrid males are sterile, the lines can be rescued by backcrossing the females to either parental species. Resistance genes can be retained in Haemonchus hybrids, while the unwanted contortus background is removed through backcrossing to H. placei and anthelmintic selection of the progeny. Under this selection, genes involved in resistance would retain the H. contortus nucleotide sequence, while those that are not would either be H. placei or a random mixture of both, depending on the amount of backcrossing that had occurred. The first candidate gene to be tested in this system was a Haemonchus P-glycoprotein, hcpgp-1. hcpgp-1 was amplified, cloned and sequenced from H. contortus and H. placei. Two restriction sites were then identified in the sequenced product; one specific to H. contortus hcpgp-1 and the other found only in the H. placei gene. These genes were identified from macrocyclic lactone selected and non-selected worms by restricting PCR products from individual worms. Fitted occurrence of the H. contortus allele was 49% of unselected worms and 69% of macrocyclic lactone selected worms. The probability of this percentage occurring by chance was P = 0.006. Thus macrocyclic lactone selection was acting to increase the percentage of hcpgp-1 from macrocyclic-lactone-resistant CAVRS.

Co-purification of Pea and Bean Leaf Soluble Auxin-binding Proteins with Ribulose-1,5-Bisphosphate Carboxylase.

Soluble auxin-binding proteins (ABPs) were purified to constant specific activity from bean and pea leaves by a procedure involving (NH(4))(2)SO(4) fractionation, anion exchange chromatography and gel filtration. Pea and bean ABPs exactly co-purify with ribulose-1,5-bisphosphate carboxylase (RuBPCase) in a variety of chromatographic separation procedures. The subunit compositions, electrophoretic purities and indole-3-acetic acid (IAA)-binding stoichiometries of the purified ABPs provide further evidence for the identity of RuBPCase and ABP. Pea ABP and bean ABP have dissociation constants for IAA of 0.8 and 1.3 micromolar, respectively, as determined by an (NH(4))(2)SO(4) precipitation assay for IAA-binding to insolubilized ABP. IAA can bind to soluble bean and pea ABP (RuBPCase) as determined by equilibrium dialysis with affinities and stoichiometries similar to those determined for insolubilized ABP.

Ligand Specificity of Bean Leaf Soluble Auxin-binding Protein.

The soluble bean leaf auxin-binding protein (ABP) has a high affinity for a range of auxins including indole-3-acetic acid (IAA), alpha-napthaleneacetic acid, phenylacetic acid, 2,4,5-trichlorophenoxyacetic acid, and structurally related auxins. A large number of nonauxin compounds that are nevertheless structurally related to auxins do not displace IAA from bean ABP. Bean ABP has a high affinity for auxin transport inhibitors and antiauxins. The specificity of pea ABP for representative auxins is similar to that found for bean ABP. The bean ABP auxin binding site is similar to the corn endoplasmic reticulum auxin-binding sites in specificity for auxins and sensitivity to thiol reagents and azide. Qualitative similarities between the ligand specificity of bean ABP and the specificity of auxin-induced bean leaf hyponasty provide further evidence, albeit circumstantial, that ABP (ribulose 1,5-bisphosphate carboxylase) can bind auxins in vivo. The high incidence of ABP in bean leaves and the high affinity of this protein for auxins and auxin transport inhibitors suggest possible functions for ABP in auxin transport and/or auxin sequestration.