Restriction fragment length patterns of DNA from parasitic nematodes of sheep.

High molecular weight DNA obtained from sheep parasitic nematodes Haemonchus contortus, Ostertagia circumcincta and Trichostrongylus colubriformis, was digested with various restriction endonucleases. Digestion with Eco R1 produced the most informative pattern of repeat sequence bands. H contortus adult or larval DNA produced bands of 2.7, 3.0 and 1.4 kb. O circumcincta adult or larval DNA had common 2.7 and 1.4 kb bands with adult specific bands of 2.2 and 0.9 kb and a larval specific 2.08 kb band. T colubriformis adults or larval DNA produced 2.7, 1.4 and 0.79 kb bands. These preliminary results show that restriction patterns of repeat sequence DNA may be useful for the identification of various trichostrongylid species parasitic for sheep.

Monoclonal antibody against sheep kappa light chain.

The production of a monoclonal antibody specific for sheep kappa light chain protein is described. The monoclonal antibody was designated McM11 and its specificity was verified using western blots of sheep IgG and slides of efferent lymph cells. The specificity of McM11 was confirmed by specific recognition of fusion proteins expressed by recombinant phage containing sheep kappa cDNAs. N terminal sequence of the light chain recognized by McM11 showed homology to kappa type light chains. McM11, together with McM6, a lambda specific monoclonal antibody, was shown by two color FACS analysis of sheep blood lymphocytes to recognize all sheep light chains.

Isolation and sequence of sheep Ig H and L chain cDNA.

Sheep lymphocyte poly(A+) RNA was used as a template for the enzymatic synthesis of cDNA before cloning into the expression vector lambda gt11. Screening of the cDNA library with mAb probes resulted in the isolation of two recombinant phages containing Ig coding sequences of 704 bp and 925 bp. These were inserted into the EcoRI site of pUC18 and named pSLC (sheep Ig L chain) and pSHC (sheep Ig H chain). The insert in pSLC revealed sequence homology by using GenBank to lambda L chain and pSHC revealed sequence homology to IgG sequences from various species. The L chain cDNA contained the full translation sequence and 5' and 3' nontranslating region while the H chain cDNA coded for the secreted form of IgG1 and lacked sequences upstream from the C region. The derived amino acid sequences showed significant homology with various Ig sequences already described for human, mouse, rabbit, pig, and chicken but the degree of homology showed no consistency with established phylogenetic relationships.

Isolation of a Vicia faba metallothionein-like gene: expression in foliar trichomes.

Animal metallothioneins (MTs) are cysteine-rich, low-molecular-weight proteins that bind to heavy metals and are believed to play a role in their metabolism and detoxification. Genes encoding MT-like proteins have been isolated in a number of plants although their function remains to be elucidated. We describe the isolation and characterization of a bean cDNA encoding an MT-like protein. The bean gene, called MT, was isolated as a result of a differential screen for genes that are expressed in leaves but not in the most common cell type, the mesophyll cell. MT contained two regions with abundant cysteines and sequence comparison found that MT had greatest homology to MT-like subtype 2 from other plant species. Northern blot analysis demonstrated that MT was expressed in the left, stem and flower, at very low levels in roots and was not detectable in mesophyll protoplasts. MT transcript levels were not significantly affected by treatment with Cu, Zn or Cd. In the left, in situ hybridization studies demonstrated striking cell specificity with MT expression confined predominantly to trichomes. Possible explanations for the pronounced expression of MT in leaf trichomes are discussed.

Analysis of immunoglobulin light chain loci in sheep.

A sheep kappa cDNA probe was isolated, characterized by sequence analysis and shown to have significant sequence identity to other kappa light chains. This probe and a sheep lambda light chain probe were used to estimate the extent of various sheep immunoglobulin light chain gene loci by Southern blot analysis of genomic DNA. The results showed that the sheep has a single hybridizing kappa constant gene and three to five kappa V segment bands. Segregation of three polymorphic bands at the lambda C locus indicated that they were products of separate C segments. Restriction fragment pattern variations were obtained using light chain probes on various sheep breeds, but no pattern or individual band was characteristic for a particular breed.

Analysis of type 1 metallothionein cDNAs in Vicia faba.

In animals and fungi, small cysteine-rich proteins called metallothioneins (MTs) play a role in heavy metal tolerance. MT genes have been isolated in plants, but their function remains to be elucidated. We have isolated two distinct Vicia faba MT genes that belong to the type 1 group of plant MT genes in contrast to a MT gene we previously isolated that belongs to type 2. We found similarities and differences between the V. faba MT genes. The RNA expression patterns differed and this was most pronounced in roots, which contained high MT1 but very low MT2 RNA levels. Like MT2, MT1 transcript levels were not significantly affected by treatment with Cd, Cu, Fe and Zn, at least under the experimental conditions. MT RNA levels varied in leaves and stem internodes of different developmental ages, with the highest expression in the older tissue. The levels of MT RNA correlated inversely with endogenous Cd, Cu and Fe levels within different internodes, but not with a number of other metals tested (including Zn). The three bean MTs were expressed in Escherichia coli and found to bind Cd, Cu and Zn but not to Fe. The MTs were tested to determine if they differed in their ability to bind a specific metal but no significant differences in binding were observed.

Isolation and characterization of two related Arabidopsis ocs-element bZIP binding proteins.

Ocs-elements are a group of related, bipartite promoter elements which have been exploited by two distinct groups of plant pathogens, Agrobacterium and certain viruses to express genes in plants. The genes for two Arabidopsis bZIP (basic region-leucine zipper) proteins that bind to ocs-elements have been isolated and characterized. The genes, called OBF4 and OBF5, were isolated by screening an Arabidopsis genomic library with degenerate oligonucleotides complementary to the DNA-binding domains of other plant ocs-element-binding proteins. The OBF4 and OBF5 proteins show 53% amino acid identity but low DNA homology. Southern blot analysis demonstrated that each of the OBF genes is a member of a small family. OBF4 is more similar to the tobacco TGA1a and Arabidopsis TGA1 proteins, while OBF5 is more similar to the maize OBF3.1, wheat HBP1b and Arabidopsis aHBP1b proteins. The DNA-binding properties of OBF4 and OBF5 were similar although OBF5 was able to bind simultaneously to both halves of the ocs-element more efficiently than OBF4. This difference in binding to the ocs-element between two closely related proteins from the same species is potentially significant since binding to both halves of the ocs-element is a pre-requisite for in vivo transcriptional activity.

A novel phloem-specific gene is expressed preferentially in aerial portions of Vicia faba.

We have isolated a gene from bean (Vicia faba L.), called Vein1, that encodes a novel protein. The Vein1 cDNA was isolated as a result of a differential screen for genes that are expressed in leaves but not in the most common cell type, the mesophyll cell. Northern blot analysis revealed that Vein 1 transcripts are differentially expressed in the plant with expression in leaves, stems and sepals but not in petals, mesophyll cells or roots. In situ hybridization studies of stem and leaf sections indicate that the expression of Vein1 is localized to the phloem tissue. Interestingly, Vein1 was differentially expressed in stem tissue with the highest expression in the oldest internodes. The deduced Vein1 protein sequence does not share homology with any known protein sequences. The 17 kDa Vein1 protein is highly hydrophilic and contains a histidine-rich motif, where six out of seven amino acids are histidines. The function of Vein1 is unknown, although the expression patterns suggests that it may play a role in mature phloem tissue in the aerial parts of the plant.

Interactions between distinct types of DNA binding proteins enhance binding to ocs element promoter sequences.

Octopine synthase (ocs) elements are a group of promoter elements that have been exploited by plant pathogens to express genes in plants. ocs elements are components of the promoters of certain plant glutathione S-transferase genes and may function as oxidative stress response elements. Genes for ocs element binding factors (OBFs), which belong to a specific class of highly conserved, plant basic domain-leucine zipper transcription factors, have been isolated and include the Arabidopsis OBF4 and OBF5 genes. To characterize proteins that modulate the activity of the OBF proteins, we screened an Arabidopsis cDNA library with the labeled OBF4 protein and isolated OBP1 (for OBF binding protein). OBP1 contains a 51-amino acid domain that is highly conserved with two plant DNA binding proteins, which we refer to as the MOA domain. OBP1 is also a DNA binding protein and binds to the cauliflower mosaic virus 35S promoter at a site distinct from the ocs element in the 35S promoter. OBP1 specifically increased the binding of the OBF proteins to ocs element sequences, raising the possibility that interactions between these proteins are important for the activity of the 35S promoter.

Isolation of a maize bZIP protein subfamily: candidates for the ocs-element transcription factor.

Ocs-elements, a family of 20 bp DNA sequences, are components of a number of promoters active in plants. In the maize BMS cell line the dominant ocs-element binding activity is the ocs-element transcription factor complex called OTF. The isolation of cDNA clones from a BMS cDNA expression library for two bZIP (basic region-leucine zipper) proteins that bind the ocs-element sequence and are good candidates for forming at least part of OTF is described. The two ocs-element binding proteins, called OBF3.1 and OBF3.2, are closely related, with the OBF3.1 protein sharing 95.8% amino acid homology with part of the OBF3.2 protein although there were significant differences in the 3' untranslated regions. Genomic Southern blot analysis revealed a small gene family with a minimum of two OBF3 loci mapping to chromosomes 3L105 and 8L075. The OBF3.1 protein shared considerable homology with the wheat HBP1b protein (80% amino acid identity) and to a lesser extent with the tobacco TGA1aa protein. OBF3.1 like HBP1b was able to bind well to the Hex sequence but poorly to G-box/ABRE sequences. Interestingly, OBF3.1 bound eightfold more efficiently to an ocs-element sequence than TGA1a, raising the possibility that OBF3.1 and TGA1a may be distinct members of an OBF3/TGA subfamily.