Western Transfer Analysis Research Articles

TFF3 is a valuable predictive biomarker of endocrine response in metastatic breast cancer.

The stratification of breast cancer patients for endocrine therapies by oestrogen or progesterone receptor expression is effective but imperfect. The present study aims were to validate microarray studies that demonstrate TFF3 regulation by oestrogen and its association with oestrogen receptors in breast cancer, to evaluate TFF3 as a biomarker of endocrine response, and to investigate TFF3 function. Microarray data were validated by quantitative RT-PCR and northern and western transfer analyses. TFF3 was induced by oestrogen, and its induction was inhibited by antioestrogens, tamoxifen, 4-hydroxytamoxifen and fulvestrant in oestrogen-responsive breast cancer cells. The expression of TFF3 mRNA was associated with oestrogen receptor mRNA in breast tumours (Pearson's coefficient=0.762, P=0.000). Monoclonal antibodies raised against the TFF3 protein detected TFF3 by immunohistochemistry in oesophageal submucosal glands, intestinal goblet and neuroendocrine cells, Barrett's metaplasia and intestinal metaplasia. TFF3 protein expression was associated with oestrogen receptor, progesterone receptor and TFF1 expression in malignant breast cells. TFF3 is a specific and sensitive predictive biomarker of response to endocrine therapy, degree of response and duration of response in unstratified metastatic breast cancer patients (P=0.000, P=0.002 and P=0.002 respectively). Multivariate binary logistic regression analysis demonstrated that TFF3 is an independent biomarker of endocrine response and degree of response, and this was confirmed in a validation cohort. TFF3 stimulated migration and invasion of breast cancer cells. In conclusion, TFF3 expression is associated with response to endocrine therapy, and outperforms oestrogen receptor, progesterone receptor and TFF1 as an independent biomarker, possibly because it mediates the malign effects of oestrogen on invasion and metastasis.

Insulin-like Growth Factor-Dependent Proliferation and Survival of Triple-Negative Breast Cancer Cells: Implications for Therapy

Triple-negative breast cancers have a poor prognosis and are not amenable to endocrine- or HER2-targeted therapies. The prevailing view is that targeting the insulin-like growth factor (IGF) signal transduction pathway will not be beneficial for triple-negative breast cancers because their growth is not IGF-responsive. The present study investigates the importance of IGFs in the proliferation and survival of triple-negative breast cancer cells. Estrogen and progesterone receptors, HER2, type I IGF, and insulin receptors were measured by Western transfer analysis. The effects of IGF-1 on proliferation were assessed by DNA quantitation and on cell survival by poly (ADP-ribose) polymerase cleavage. The effect of IGF-1 on phosphorylation of the IGF receptors, Akt and mitogen-activated protein kinase, was measured by Western transfer analysis. Seven cell lines were identified as models of triple-negative breast cancer and shown to express IGF receptors at levels similar to those present in estrogen-responsive cell lines known to respond to IGFs. IGF-1 increased the proliferation and cell survival of all triple-negative cell lines. Proliferation was attenuated after reduction of type I IGF receptor expression. Cells that express higher levels of receptor were more sensitive to subnanomolar IGF-1 concentrations, but the magnitude of the effects was not correlated simply with the absolute amount or phosphorylation of the IGF receptors, Akt or mitogen-activated protein kinase. These results show that IGFs stimulate cell proliferation and promote cell survival in triple-negative breast cancer cells and warrant investigation of the IGF signal transduction pathway as a therapeutic target for the treatment of triple-negative breast cancer.

Abstract 1201: Role of the chemokine CXCL12 in the estrogen response of breast cancer cells

Abstract The purpose of this study was to investigate the extent to which CXCL12 mediates the effects of estrogen in estrogen-responsive breast cancer cells. Expression microarray studies have identified CXCL12 as a gene regulated consistently by estrogen in estrogen-responsive breast cancer cells in our own and other laboratories and it has been reported that CXCL12 mediates the proliferative effects of estrogen. CXCL12 activates two seven transmembrane G protein-coupled receptors, CXCR4 and CXCR7, that transducer signals via heterotrimeric G-proteins. CXCR4 has long been accepted, whereas CXCR7 was identified more recently as a potential CXCL12 receptor. Methods: Expression of CXCL12, CXCR4 and CXCR7 was measured in five estrogen-responsive and five estrogen-nonresponsive breast cancer cell lines by qRT-PCR and western transfer analysis. The effects of CXCL12 on proliferation of the cells were assessed by DNA quantification and on migration in a modified Boyden chamber assay. The ability of CXCL12 to activate the downstream signalling molecules, MAPK and Akt, was measured by western transfer analysis with a phospho-specific antibodies. The motogenic responses of different breast cancer cell lines to CXCL12 were measured in modified Boyden chamber and in vitro wounding assays. The relative importance of CXCR4 in the CXCL12 response was probed with a specific inhibitor of CXCR4 receptor. Results: The expression of CXCL12, CXCR4 and CXCR7 varied greatly between the ten breast cancer cell lines analyzed. CXCL12 expression was highest in estrogen-responsive T-47D cells whereas CXCR4 and CXCR7 expression was highest in estrogen-nonresponsive MDA-MB-231 cells. CXCL12 was detected after secretion into medium conditioned by T-47D and MCF-7 breast cancer cells. CXCL12 did not stimulate the proliferation of MCF-7 or T-47D cells in the absence or presence of estrogen. CXCL12 did stimulate breast cancer cell migration in a concentration-dependent manner and cells that express high concentrations of the CXCL12 receptor were more responsive to CXCL12. CXCL12 stimulated Phosphorylation of both MAPK and Akt in estrogen-responsive and non-responsive breast cancer cells. Conclusion: This study shows that CXCL12 does not mediate the proliferative effects of estrogen on breast cancer cells. CXCL12 does however stimulate the migration of both estrogen-responsive and estrogen non-responsive breast cancer cells. CXCL12 mediates it effects in breast cancer cells by activation on both the MAPK and Akt signal transduction pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1201. doi:10.1158/1538-7445.AM2011-1201

Abstract 3175: Importance of CXCL12, CXCR4 and CXC7 in breast cancer cells

Abstract The purpose of this study is to understand the relative importance of the two chemokine receptors CXCR4 and CXCR7 in the response of estrogen-responsive and estrogen-nonresponsive breast cancer cells to the chemokine CXCL12. CXCR4 has long been accepted, whereas CXCR7 was identified more recently as a CXCL12 receptor. CXCL12 is expressed by stromal and epithelial cells in breast tumors and has been shown to be regulated by estrogen in estrogen-responsive breast cancer epithelial cells. CXCL12 activates the two seven transmembrane G protein-coupled receptors, CXCR4 and CXCR7, that transduce signals via heterotrimeric G-proteins. Methods The expression of CXCL12, CXCR4 and CXCR7 was measured in five estrogen-responsive and five estrogen-nonresponsive breast cancer cell lines by qRT-PCR and western transfer analysis. The ability of CXCL12 to activate the CXCR4 receptors was measured by western transfer analysis with a phospho-specific antibody. The motogenic responses of different breast cancer cell lines to CXCL12 were measured in modified Boyden chamber and in vitro wounding assays. The relative importance of CXCR4 in the CXCL12 response was probed with a specific inhibitor of CXCR4 receptor. Results The expression of CXCL12, CXCR4 and CXCR7 varied greatly between the ten breast cancer cell lines analyzed. CXCL12 expression was highest in estrogen-responsive T-47D cells whereas CXCR4 and CXCR7 expression was highest in estrogen-nonresponsive MDA-MB-231 cells. CXCL12 protein expression was not detected in protein extracts from breast cancer cells. CXCL12 was detected however after secretion into medium conditioned by T-47D and MCF-7 breast cancer cells. Chemotaxis assays showed that CXCL12 stimulated breast cancer cell migration in a concentration-dependent manner and cells that expressed higher concentrations of the receptors were more responsive to CXCL12. CXCR4 phosphorylation was stimulated by CXCL12 in some but not all breast cancer cells. AMD3100, a competitive inhibitor of CXCL12 binding to CXCR4, inhibited CXCL12-induced migration. Maximum inhibition of migration stimulated by 12.5 nM CXCL12 was achieved with 5 µM AMD3100. Conclusion This study shows that CXCL12 is an autocrine motogen in estrogen-responsive breast cancer cells. CXCL12 stimulates migration of both estrogen-responsive and estrogen-nonresponsive breast cancer cells. The sensitivity of the migratory response of breast cancer cells to CXCL12 depends on the relative concentrations of CXCR4 and CXCR7. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3175.

Class III β-Tubulin Is a Component of the Mitotic Spindle in Multiple Cell Types

The findings of this study show that Class III beta-tubulin is a component of the mitotic spindle in multiple cell types. Class III beta-tubulin has been widely used as a neuron-specific marker, but it has been detected also in association with breast and pancreatic cancers. In this study, we describe a novel finding of Class III beta-tubulin in a subpopulation of cells in malignant peripheral nerve sheath tumor. The findings of this study also show that Class III beta-tubulin is expressed by normal mesenchymal and epithelial cells (fibroblasts and keratinocytes), two transitional cell carcinoma cell lines, and neurofibroma Schwann cells, as shown by immunolabeling and Western transfer analysis using two different Tuj-1 antibodies that are specific for Class III beta-tubulin. The corresponding mRNA was detected using RT-PCR and whole human genome microarrays. Both antibodies localized Class III beta-tubulin to the mitotic spindle and showed a colocalization with alpha-tubulin. The immunoreaction became visible in early prophase, and the most intense immunoreaction was detected during metaphase and anaphase when microtubules were connected to the kinetochores on chromosomes. Class III beta-tubulin-specific immunoreaction lasted to the point when the midbody of cytokinesis became detectable.

Tight Junction Proteins and Perineurial Cells in Neurofibromas

Cutaneous neurofibromas consist of axonal processes, Schwann cells, fibroblasts, perineurial cells, mast cells, and abundant extracellular matrix. The distribution and role of perineurial cells in neurofibromas has been uncertain, partly because there has not been a specific immunohistochemical marker for perineurial cells. In this study, tight junctions (TJs) of 16 neurofibromas from 12 patients with neurofibromatosis type 1 (NF1) were analyzed using electron microscopy, immunohistochemistry, and Western transfer analysis. Cell-cell contacts with typical ultrastructural morphology of TJs were seen between adjacent perineurial cells surrounding the small nerves and between contacting perineurial cell processes embedded in tumor stroma. Immunohistochemistry showed expression of claudin-1, claudin-3, and ZO-1 in the intercellular junctions of a subpopulation of tumor cells. Occludin was present mainly in perineurium and claudin-5 localized to the blood vessels. Double immunolabelings were used to identify the cell types expressing claudin-1. The results showed that claudin-1 positive cells were also positive for type IV collagen and epithelial membrane antigen but not for S-100 protein. This labeling pattern is consistent with perineurial cell phenotype. Using claudin-1 as a marker, our results showed that clusters of perineurial cells are distributed around the rudimentary nerves within cutaneous neurofibromas and at the periphery of some neurofibromas.

The Diurnal Rhythm of the Cytoprotective Human Trefoil Protein TFF2 Is Reduced by Factors Associated with Gastric Mucosal Damage: Ageing, Helicobacter pylori Infection, and Sleep Deprivation

To determine if the normal TFF2 diurnal rhythm is disrupted in those with increased risk of gastric morbidity. Trefoil proteins protect the gastrointestinal mucosa from damage and aid its repair. TFF2 is considered the major cytoprotective gastric trefoil protein. There is a marked circadian variation in gastric luminal TFF2 in young healthy volunteers with peak levels present during the night. Gastric juice was aspirated at two hourly intervals over a 24-h period via a nasogastric tube. TFF2 was measured by quantitative western transfer analysis. Helicobacter pylori (H. pylori) status was measured by C13 urea breath test and by serology. The effects of H. pylori infection, sleep deprivation, and ageing, which cause increased gastric morbidity, on the TFF2 circadian rhythm were tested. H. pylori infection attenuated the increase in TFF2 that occurs during the night. The TFF2 diurnal rhythm was reduced in older people and both the TFF2 level reached and the time at which the maximum TFF2 concentration occurs were associated inversely with age (p < 0.005). Sleep deprivation delayed the normal night time increase in gastric TFF2 and resulted in an overall reduction in TFF2 secretion. H. pylori infection, ageing, and sleep deprivation cause a reduction in the TFF2 diurnal rhythm. The demonstration that the TFF2 rhythm is impaired in cohorts of individuals known to suffer gastric symptoms suggests that interventions to restore the normal TFF2 rhythm in those with poor mucosal protection could reduce morbidity.

Active Kinase Proteome Screening Reveals Novel Signal Complexity in Cardiomyopathy

Recent advances in the characterization of the phosphoproteome have been limited to measuring phosphorylation statuses, which imply but do not measure protein kinase activity directly. As such, the ability to screen, compare, and define multiple protein enzymatic activities across divergent samples remains a daunting challenge in proteomics. Here, we describe a gel-based kinase assay coupled to MS identification as an approach to map global kinase activity and assign pathway architecture to specified biologic contexts. We demonstrate the utility of this method as a platform for the comparison of proteomes based on differences in both kinase activities and for use in the de novo substrate identification for individual kinases. This approach allowed us to map the signal perturbations in the post-natal heart that were associated with activation of a myopathic cascade as mediated by the mitogen-activated protein kinase MKK6 and established the novel observation that MKK6 promotes the development of cardiomyopathy through multiple substrate interactions.

The trefoil protein TFF1 is bound to MUC5AC in human gastric mucosa.

The trefoil protein TFF1 is expressed principally in the superficial cells of the gastric mucosa. It is a small protein and forms homo- and hetero-dimers via a disulphide bond through Cys58 which is located three amino acids from the C terminus. TFF1 is co-expressed with the secreted mucin MUC5AC in superficial cells of the gastric mucosa suggesting that it could be involved in the packaging or function of gastric mucus. We have previously shown that TFF1 co-sediments with mucin glycoproteins on caesium chloride gradients. To extend this observation we have now used gel filtration under physiological conditions, immunoprecipitation and Western transfer analysis to characterise the interaction of TFF1 with gastric mucin glycoproteins. We show that TFF1 co-elutes with MUC5AC but not MUC6 on gel filtration and that immunoprecipitation and Western transfer analysis confirms that TFF1 interacts with MUC5AC. We also demonstrate that the TFF1 dimer is the predominant molecular form bound to MUC5AC. Salt and chelators of divalent cations such as EDTA and EGTA disrupted the TFF1- MUC5AC interaction and increased the degradation of MUC5AC, whereas calcium increased the amount of TFF1 bound to MUC5AC. These data support the contention that TFF1 is pivotal in the packaging and function of human gastric mucosa.

NF1 tumor suppressor protein and mRNA in skeletal tissues of developing and adult normal mouse and NF1-deficient embryos.

NF1 is a heritable disease with multiple osseous lesions. The expression of the NF1 gene was studied in embryonic and adult rodent skeleton and in NF1-deficient embryos. The NF1 gene was expressed intensely in the cartilage and the periosteum. Impaired NF1 expression may lead to inappropriate development and dynamics of bones and ultimately to the osseous manifestations of the disease. Neurofibromatosis type 1 is caused by mutations in the NF1 gene encoding the Ras GTPase activating protein (Ras-GAP) neurofibromin. Skeletal ailments such as short stature, kyphoscoliosis, and tibial bowing and pseudarthrosis are common osseous manifestations of NF1. These symptoms are congenital, implying a role for neurofibromin in proper bone growth. However, little is known about its expression in skeletal tissues during their development. The expression of the NF1 gene was studied in normal and NF1+/- mouse fetuses at embryonic days 12.5-15.5 and in skeletal tissues of adult mice and rats. In situ hybridization, immunohistochemistry, and Western blot analysis were used to identify the NF1 gene expression profile. NF1 mRNA and protein were elevated in resting, maturation, and hypertrophic chondrocytes at the growth plate. Parallel studies on NF1+/- embryos showed expression patterns identical to wildtype. The periosteum, including osteoblasts and osteoclasts, and osteocytes of the cortical bone of adult mice were also intensely labeled for NF1 protein and mRNA. Western transfer analysis detected NF1 protein in the respective rat tissues. Phosphorylation of p42 and p44 MAP kinases, the downstream consequence of Ras activation, was elevated in hypertrophic chondrocytes of NF1+/- embryos. The results suggest that neurofibromin may act as a Ras-GAP in skeletal cells to attenuate Ras transduced growth signals and thus play a role during ossification and dynamics of bone. Loss of NF1 function may therefore lead to dysplastic bone growth, thereby causing the debilitating osseous symptoms of NF1.

Functional expression of NF1 tumor suppressor protein: association with keratin intermediate filaments during the early development of human epidermis.

BackgroundNF1 refers to type 1 neurofibromatosis syndrome, which has been linked with mutations of the large NF1 gene. NF1 tumor suppressor protein, neurofibromin, has been shown to regulate ras: the NF1 protein contains a GTPase activating protein (GAP) related domain which functions as p21rasGAP. Our studies have previously demonstrated that the NF1 protein forms a high affinity association with cytokeratin 14 during the formation of desmosomes and hemidesmosomes in cultured keratinocytes.MethodsThe expression of NF1 protein was studied in developing human epidermis using western transfer analysis, indirect immunofluorescence, confocal laser scanning microscopy, immunoelectron microscopy, and in situ hybridization.ResultsThe expression of NF1 protein was noted to be highly elevated in the periderm at 8 weeks estimated gestational age (EGA) and in the basal cells at 8–14 weeks EGA. During this period, NF1 protein was associated with cytokeratin filaments terminating to desmosomes and hemidesmosomes. NF1 protein did not display colocalization with α-tubulin or actin of the cytoskeleton, or with adherens junction proteins.ConclusionsThese results depict an early fetal period when the NF1 tumor suppressor is abundantly expressed in epidermis and associated with cytokeratin filaments. This period is characterized by the initiation of differentiation of the basal cells, maturation of the basement membrane zone as well as accentuated formation of selected cellular junctions. NF1 tumor suppressor may function in the regulation of epidermal histogenesis via controlling the organization of the keratin cytoskeleton during the assembly of desmosomes and hemidesmosomes.

Altered Calcium-Mediated Cell Signaling in Keratinocytes Cultured from Patients with Neurofibromatosis Type 1

Capacitative calcium entry and calcium wave propagation were studied in keratinocytes cultured from control persons and patients with type 1 neurofibromatosis. The cells were stimulated mechanically in the presence of inhibitors of gap-junctional or ATP-mediated communication to determine which pathways are operative in Ca(2+) signaling between these cells. Keratinocytes cultured from patients with type 1 neurofibromatosis (NF1) had a tendency to form cultures with markedly altered calcium-related signaling characteristics. Specifically, the resting Ca(2+) levels, intracellular Ca(2+) stores, capacitative calcium influx, and gap-junctional signal transduction were defective in NF1 keratinocytes. Western transfer analysis revealed apparently equal connexin 43 protein levels in normal control and in NF1 keratinocytes. Indirect immunofluorescence, however, demonstrated that connexin 43 was relatively evenly distributed in NF1 cells and did not form typical gap-junctional plaques between keratinocytes. Furthermore, the speed of the calcium wave was reduced in NF1 cells compared to normal keratinocytes. The results demonstrate that keratinocytes cultured from patients with NF1 display altered calcium-mediated signaling between cells.

Dramatic diurnal variation in the concentration of the human trefoil peptide TFF2 in gastric juice

BACKGROUNDTFF2, a member of the trefoil factor family of proteins, is a glycosylated protein of 106 amino acids. It is secreted by gastric antral and pyloric glands and by Brunner's...

The human trefoil peptide, TFF1, is present in different molecular forms that are intimately associated with mucus in normal stomach

BACKGROUNDTFF1 is a 6.5 kDa secreted protein that is expressed predominantly in normal gastric mucosa. It is coexpressed with mucins and it can form dimers via a free carboxy terminal...

Membrane expression of HLA-Cw4 free chains in activated T cells of transgenic mice.

Transgenic mice were produced in which human HLA-Cw4 is stably integrated, behaves as a single Mendelian trait, and, being under the transcriptional control of human CD2, is selectively and efficiently expressed in T lymphocytes. These mice were used as a model system to determine whether HLA-type C molecules can be exposed on the surface of activated lymphocytes as free heavy chains, non-associated with beta2-microglobulin (beta2m). In our transgenic mice we could identify HLA-Cw4 molecules either as free chains or as beta2m-associated molecules by the use of monoclonal antibodies specific for either conformation of HLA class I and nonreactive to mouse H2 molecules. Resting mouse lymphocytes were shown by western transfer analysis to contain sizeable amounts of HLA-Cw4 free chains, but they exposed on their surface HLA-Cw4 only in association with beta2m, as indicated by flow cytometric measurements. Conversely, where the content of total HLA-Cw4 was increased, lectin-activated mouse lymphocytes exposed on their outer cell membrane HLA-Cw4 molecules in both conformations, namely, also as free heavy chains. Isoelectrofocusing analysis confirmed the presence of both HLA-Cw4 molecular conformations in activated T cells and indicated that HLA-Cw4 heavy chains can bind to mouse beta2m with the same low affinity displayed for human beta2m. The results of our experiments led us to conclude that (1) association with beta2m is not necessary for the exposure of HLA-C on the surface of activated T lymphocytes and (2) cell activation affects the balance between the two conformational forms of HLA-C.

Characterization of in vivo salivary-derived enamel pellicle

Salivary proteins and glycoproteins that participate in the formation of 2-h in vivo enamel pellicle were determined utilizing polyacrylamide gel electrophoresis [sodium dodecyl sulphate (SDS)-PAGE and anionic PAGE]/Western transfer analyses, and specific radiolabelling/SDS-PAGE fluorography. The sensitivity of these methods permitted the identification of individual members of different salivary protein families. The major components of this pellicle were salivary α-amylase, cysteine-containing phosphoprotein (CCP or cystatins), salivary mucin and sIgA. Glycosylated amylase was present in larger quantity than the non-glycosylated species. Only CCPI (cystatin SA-I) of the cysteine-containing phosphoprotein family was identified. The higher molecular-weight salivary mucin (MG1), but not the lower molecular-weight species (MG2), was detected. These results extend earlier observations regarding the selective nature of salivary protein adsorption to enamel surface by demonstrating that only specific members of salivary protein families are involved in 2-h in vivo enamel pellicle formation. The findings also suggest that individual family members may have different functions in the mouth.

Immune properties of glucosyltransferases from S. sobrinus.

GTF activity was separated into water-insoluble (GTF-I) and water-soluble (GTF-S) polyglucan-synthesizing enzymes. Each preparation demonstrated a single band on 6% SDS PAGE. Only water-insoluble or water-soluble polyglucan was synthesized by the respective enzyme preparation. Rats were immunized, on Days 1 and 14, with either GTF-I or GTF-S in adjuvant. Animals were bled 13, 35 and 54 days after the initial immunization. Individual antisera were tested against either the GTF-I or the GTF-S for inhibition of radioactive glucose incorporation into glucan, and in gel diffusion, and by Western transfer analyses. The respective antisera reacted with the homologous, but not the heterologous enzyme in gel diffusion and Western transfer. GTF-I activity was not inhibited by antibody to GTF-S, but antibody to GTF-I inhibited GTF-I by 68%. GTF-S was inhibited by more than 60% by each of 3 anti-GTF-S sera. Only one anti-GTF-I serum inhibited GTF-S at as much as a modest 30% level. These data support the antigenic and functional distinctiveness of the GTF enzymes of S. sobrinus 6715.

Morphogenetic expression of Bacteroides nodosus fimbriae in Pseudomonas aeruginosa.

Type 4 fimbriae are found in a range of pathogenic bacteria, including Bacteroides nodosus, Moraxella bovis, Neisseria gonorrhoeae, and Pseudomonas aeruginosa. The structural subunits of these fimbriae all contain a highly conserved hydrophobic amino-terminal sequence preceding a variable hydrophilic carboxy-terminal region. We show here that recombinant P. aeruginosa cells containing the B. nodosus fimbrial subunit gene under the control of a strong promoter (pL, from bacteriophage lambda) produced large amounts of fimbriae that were structurally and antigenically indistinguishable from those produced by B. nodosus. This was demonstrated by fimbrial isolation and purification, electrophoretic and Western transfer analyses, and immunogold labeling and electron microscopy. These results suggest that type 4 fimbriated bacteria use a common mechanism for fimbrial assembly and that the structural subunits are interchangeable, thereby providing a basis for the development of multivalent vaccines.

Molecular cloning of the fimbrial subunit gene from a benign type B isolate ofBacteroides nodosus

The fimbrial subunit gene from the benign type B Bacteroides nodosus isolate AC/6 was cloned into the SphI site of the multicopy vector plasmid pUC19. Five Escherichia coli recombinants that were positive in a colony immunoassay were shown, by Western transfer analysis, to produce an immunologically cross-reacting protein of identical molecular size to fimbrial subunits prepared from B. nodosus AC/6. Restriction endonuclease analysis showed that 4 of the recombinant plasmids carried a 6.7 kb SphI fragment. Recloning experiments showed that the fimbrial subunit gene was located within a 2.5 kb EcoRI-SphI fragment and that there was a PstI site located within the structural gene or its regulatory region. These recombinant clones will prove useful for the construction of a multivalent recombinant vaccine for the control of ovine footrot.

Molecular cloning of the fimbrial subunit gene from a benign type B isolate of Bacteroides nodosus

The fimbrial subunit gene from the benign type BBacteroides nodosus isolate AC/6 was cloned into theSphI site of the multicopy vector plasmid pUC19. FiveEscherichia coli recombinants that were positive in a colony immunoassay were shown, by Western transfer analysis, to produce an immunologically cross-reacting protein of identical molecular size to fimbrial subunits prepared fromB. nodosus AC/6. Restriction endonuclease analysis showed that 4 of the recombinant plasmids carried a 6.7 kbSphI fragment. Recloning experiments showed that the fimbrial subunit gene was located within a 2.5 kbEcoRI-SphI fragment and that there was aPstI site located within the structural gene or its regulatory region. These recombinant clones will prove useful for the construction of a multivalent recombinant vaccine for the control of ovine footrot.