A comparative study of the quality of DNA obtained from fresh frozen and formalin-fixed decalcified paraffin-embedded bone marrow trephine biopsy specimens using two different methods.

BACKGROUND: Given its prognostic value, there is renewed interest in molecular staging in non-Hodgkin's lymphoma (NHL) using immunoglobulin heavy and light chain (IgH, IgL) gene rearrangements. AIMS: To compare the efficiency of DNA amplification from fresh frozen and formalin-fixed decalcified paraffin-embedded (FFDPE) bone marrow trephines for use in molecular staging using two methods. METHODS: After manually extracting DNA from 13 FFDPE and 14 fresh frozen trephine biopsy specimens, two methods were used to test for amplifiability: use of the amplification control master mix supplied in the In Vivo Scribe immunoglobulin heavy chain (IgH) clonality kit, which creates 5 amplicons between 96-600 base pairs (bp); and real-time amplification of the beta-globin gene. RESULTS: Using the first method, the mean maximum length of amplicons generated from FFDPE trephines was statistically lower at 300 bp compared to fresh frozen samples, all of which generated amplicons up to 600 bp in size (p<0.001). Real-time amplification of the beta-globin gene showed that the mean crossing threshold of fresh frozen samples was statistically lower than that of FFDPE samples (23.48 (95% CI 22.47 to 24.48) vs 33.64 (95% CI 32.15 to 35.12); p<0.001). CONCLUSIONS: Although amplifiable DNA can be extracted from both fresh-frozen and FFDPE trephine samples for IgH/IgL analysis, freshly frozen specimens are superior as a source of template DNA, especially for higher base pair PCR products.

CD97 is a processed, seven-transmembrane, heterodimeric receptor associated with inflammation.

CD97 is a receptor that spans the membrane seven times, a defining feature of G protein-coupled receptors. CD97 is predominantly expressed in leukocytes, but the function and accurate protein structure of this receptor have not been described. We show here that CD97 has the novel property among G protein-coupled receptors characterized to date of being processed intracellularly in either the endoplasmic reticulum or early Golgi from a proprotein into a noncovalently associated two-subunit structure that becomes expressed on the cell surface and is composed of a large extracellular protein (CD97alpha) and a seven-membrane spanning protein (CD97beta). CD97beta is part of an evolutionarily conserved subfamily of four proteins, including two Caenorhabditis elegans proteins of as yet unknown function, which is distinct from but most closely related to the glucagon receptor family. CD97alpha exists in three alternatively spliced isoforms that contain between three and five epidermal growth factor (EGF)-like repeats that are related to the calcium binding EGF-like repeats in the microfibril protein fibrillin. Leukocytes strongly positive for CD97 are concentrated at sites of inflammation relative to CD97 expression in normal lymphoid tissues. Soluble CD97alpha was found in body fluids from inflamed tissues, suggesting that a functional consequence of the CD97 heterodimeric structure is the stable existence of CD97alpha in a cellfree form. CD97 appears to be a multifunctional protein that may play a signal transduction role associated with the establishment or development of an inflammatory process.

Heterologous exopolysaccharide production in Rhizobium sp. strain NGR234 and consequences for nodule development.

Rhizobium sp. strain NGR234 produces large amounts of acidic exopolysaccharide. Mutants that fail to synthesize this exopolysaccharide are also unable to nodulate the host plant Leucaena leucocephala. A hybrid strain of Rhizobium sp. strain NGR234 containing exo genes from Rhizobium meliloti was constructed. The background genetics and nod genes of Rhizobium sp. strain NGR234 are retained, but the cluster of genes involved in exopolysaccharide biosynthesis was deleted. These exo genes were replaced with genes required for the synthesis of succinoglycan exopolysaccharide from R. meliloti. As a result of the genetic manipulation, the ability of these hybrids to synthesize exopolysaccharide was restored, but the structure was that of succinoglycan and not that of Rhizobium sp. strain NGR234. The replacement genes were contained on a cosmid which encoded the entire known R. meliloti exo gene cluster, with the exception of exoB. Cosmids containing smaller portions of this exo gene cluster did not restore exopolysaccharide production. The presence of succinoglycan was indicated by staining with the fluorescent dye Calcofluor, proton nuclear magnetic resonance spectroscopy, and monosaccharide analysis. Although an NGR234 exoY mutant containing the R. meliloti exo genes produced multimers of the succinoglycan repeat unit, as does the wild-type R. meliloti, the deletion mutant of Rhizobium sp. strain NGR234 containing the R. meliloti exo genes produced only the monomer. The deletion mutant therefore appeared to lack a function that affects the multiplicity of succinoglycan produced in the Rhizobium sp. strain NGR234 background. Although these hybrid strains produced succinoglycan, they were still able to induce the development of an organized nodule structure on L. leucocephala. The resulting nodules did not fix nitrogen, but they did contain infection threads and bacteroids within plant cells. This clearly demonstrated that a heterologous acidic exopolysaccharide structure was sufficient to enable nodule development to proceed beyond the developmental barrier imposed on mutants of Rhizobium sp. strain NGR234 that are unable to synthesize any acidic exopolysaccharide.

Functional and evolutionary relatedness of genes for exopolysaccharide synthesis in Rhizobium meliloti and Rhizobium sp. strain NGR234.

Rhizobium meliloti SU47 and Rhizobium sp. strain NGR234 produce distinct exopolysaccharides that have some similarities in structure. R. meliloti has a narrow host range, whereas Rhizobium strain NGR234 has a very broad host range. In cross-species complementation and hybridization experiments, we found that several of the genes required for the production of the two polysaccharides were functionally interchangeable and similar in evolutionary origin. NGR234 exoC and exoY corresponded to R. meliloti exoB and exoF, respectively. NGR234 exoD was found to be an operon that included genes equivalent to exoM, exoA, and exoL in R. meliloti. Complementation of R. meliloti exoP, -N, and -G by NGR234 R'3222 indicated that additional equivalent genes remain to be found on the R-prime. We were not able to complement NGR234 exoB with R. meliloti DNA. In addition to functional and evolutionary equivalence of individual genes, the general organization of the exo regions was similar between the two species. It is likely that the same ancestral genes were used in the evolution of both exopolysaccharide biosynthetic pathways and probably of pathways in other species as well.

Exopolysaccharide production in Rhizobium and its role in invasion.

A complex interaction between rhizobia and specific legume plants results in the formation of nitrogen-fixing root nodules. The necessity for signal exchange and a chemically based recognition system between the symbiotic partners has been appreciated for some time, but the details are only gradually being elucidated. The two basic nodule ontogenies exhibit different requirements for Rhizobium exopolysaccharides. These surface exopolysaccharide molecules of Rhizobium are synthesized at a membrane complex, which is regulated by both transcriptional and post-translational controls. The acidic exopolysaccharide probably plays both a passive and an active role during the invasion process.

Two genes that regulate exopolysaccharide production in Rhizobium sp. strain NGR234: DNA sequences and resultant phenotypes.

Two closely linked genes involved in the regulation of exopolysaccharide (EPS) production in Rhizobium sp. strain NGR234, exoX and exoY, were sequenced, and their corresponding phenotypes were investigated. Inhibition of EPS synthesis occurred in wild-type strains when extra copies of exoX were introduced, but only when exoY had been deleted or mutated or was present at a lower copy number. Normal EPS synthesis occurred in Rhizobium sp. when both exoX and exoY were introduced on the same replicon. Surprisingly, the presence of multiple copies of exoY in exoY:: Tn5 mutants of NGR234 adversely affected cellular growth. This was apparent when exoY was introduced into exoY mutants on IncP1 vectors, where the copy number was approximately 10, but was not apparent when present on much larger R-prime plasmids with lower copy numbers (approximately 3 per cell). Multiple copies of exoX did not adversely affect cellular growth of any strain. The exoX gene appeared analogous, in size and phenotype, to a previously described Rhizobium leguminosarum biovar phaseoli EPS gene, psi (D. Borthakur and A.W.B. Johnston, Mol. Gen. Genet. 207:149-154, 1987), and the deduced ExoX and Psi shared strikingly similar secondary structures. Despite this, ExoX and Psi showed little homology at the primary amino acid level, except for a central region of 18 amino acids. The interaction of ExoX and ExoY could form the basis of a sensitive regulatory system for EPS acids. The interaction of ExoX and ExoY could form the basis of a sensitive regulatory system for EPS biosynthesis. The presence of a multicopy exoX in Rhizobium meliloti and R. fredii similarly abolished EPS biosynthesis in these species.