No abstract

Establishment of a nitrogen-fixing root nodule is accompanied by a developmentally regulated expression of nodulin genes, only some of which, the so-called early nodulin genes, are expressed in stages preceding actual nitrogen fixation. We have isolated soybean cDNA clones representing early nodulin genes and have studied clone pENOD2 in detail. The cDNA insert of this clone hybridizes to nodulespecific RNA of 1200 nucleotides in length. The RNA that was hybrid-selected by the cloned ENOD2 DNA was in vitro translated to produce two nodulins with an apparent Mr of 75,000, the N-75 nodulins. These two nodulins differ slightly in charge and one does not contain methionine. The amino acid sequence deduced from the DNA sequence shows that proline accounts for 45% of the 240 residues in these nodulins and the sequence contains at least 20 repeating heptapeptide units. The amino acid composition of none of the (hydroxy)proline-rich (glyco)proteins described in plants resembles the composition of the N-75 nodulins, especially with respect to the high glutamic acid and the low serine content. This suggests that the N-75 nodulins belong to a hitherto unidentified class of presumably structural proteins. The genes encoding the N-75 nodulins were found to be expressed in nodule-like structures devoid of intracellular bacteria and infection threads, indicating that these nodulins do not function in the infection process but more likely function in nodule morphogenesis.The formation of nitrogen-fixing nodules on the roots of leguminous plants induced by bacteria of the genera Rhizobium and Bradyrhizobium involves the specific expression of a number of plant genes called nodulin genes (1-3). In a description of nodule development, Vincent (4) distinguishes between three stages in nodule development denoted as "preinfection", "infection and nodule formation", and "nodule function". In the preinfection stage, the Rhizobium bacteria recognize their host plants and attach to the root hairs, an event that is followed by root hair curling. At the moment, nothing is known about specific plant genes that are involved in this stage. In the next stage, the bacteria enter the roots by infection threads while concomitantly the dedifferentiation of some cortical cells results in the formation of meristems. The infection threads grow toward the meristematic cells; bacteria are released into the cytoplasm of about half of these cells and develop into bacteroids. In the final stage, further differentiation of nodule cells occurs leading up to a nitrogen-fixing nodule. Most studies on the expression of nodulin genes so far have been confined to the final stage of root nodule development. But the steps involved in root nodule formation show that major decisions determining the development of a root nodule are made in the stages preceding the establishment of a nitrogen-fixing nodule. We have shown (5) that nodulin genes are differentially expressed during development and that in pea at least two nodulin genes are transcribed in the seco...

The expression of plant genes involved in the pea‐Rhizobium symbiosis was studied by analysing mRNA from root nodules. The RNA was translated in vitro and the translation products were separated by two‐dimensional gel electrophoresis. The results show differential expression of nodulin genes during root nodule development. One gene encoding N‐40′ is expressed at a significant level 5 days before the leghemoglobin genes. Most other nodulin genes are expressed more or less concomitantly with the leghemoglobin genes whereas the N‐21 mRNA is only present late during the development. In the development of ineffective root nodules induced by infection with different nod+fix− mutants of R. leguminosarum all nodulin genes are expressed except for the N‐21 gene. The results suggest that neither bacteroid development, heme excretion nor nitrogen fixation are essential for the induction of nodulin gene expression in the host plant. Further, it appears that the amount of leghemoglobin in ineffective nodules is regulated at a post‐transcriptional level.

In vitro translation products of total RNA isolated from soybean nodules at successive stages of nodule development were analyzed by two-dimensional gel electrophoresis. In that way the occurrence of over 20 mRNAs specifically transcribed from nodulin genes was detected. The nodulin genes could be divided into two classes according to the time of expression during nodule development. Class A comprises at least 4 nodulin mRNAs which are found when a globular meristem is present in the root cortex. These class A nodulin genes have a transient expression. Class B nodulin genes are expressed when the formation of a nodule structure has been completed. Bradyrhizobium japonicum nod (+) fix(-)mutants, with large deletions spanning the nif H,DK region, still induced nodules showing normal expression of all nodulin genes, indicating that the nif H,DK region is not involved in the induction of nodulin genes. In nodules induced by Bradyrhizobium japonicum nod (+) fix(-)mutant HS124 the bacteria are rarely released from the infection thread and the few infected cells appear to be collapsed. All class A and class B nodulin genes are expressed in HS124 nodules with the exception of 5 class B genes.

Non-islet-cell tumour-induced hypoglycaemia (NICTH) is, in most cases, attributable to tumour production of insulin-like growth factor II (IGF-II). Tumour-derived IGF-II has a higher than normal molecular weight (big 'IGF-II') and an impaired ability to form the normal ternary 150 kD complex with IGF binding protein-3 (IGFBP-3) and the acid-labile subunit (ALS). Consequently, tumoral IGF-II circulates mainly in smaller binary complexes which have a higher bioavailability than the ternary complex. We had the opportunity to analyze IGFs and IGF-related factors in both pre- and post-operative blood, tumour tissue and tumour cyst fluid from a patient with a disseminated haemangiopericytoma and severe hypoglycaemia. In addition, the effect of serum and tumour cyst fluid on autophosphorylation of the insulin receptor was examined. Patient serum contained low levels of IGF-I, IGFBP-3 and ALS, while the concentrations of IGFBP-2 and IGFBP-6 were markedly elevated. The total level of circulating IGF-II was within the normal range, but Biogel P-60 gel filtration of patient serum revealed that 77% of the IGF-II was present in high molecular weight forms (normal: 10-15%), which decreased to 53% after partial removal of the tumour. Most of the IGF-II immunoreactivity in pre- and post-operative patient serum was associated with 50-60 kD complexes with only a minimal contribution (<10%) from the 150 kD complex. Tumour cyst fluid contained excessive amounts of both big IGF-II and IGFBP-6. Northern blot analysis of total mRNA isolated from the tumour demonstrated high expression of the IGF-II gene and abundant 1.1 kb IGFBP-6 transcript, while the genes encoding IGFBP-3, -4 and -5 were only weakly expressed and mRNA of IGFBP-1, -2 and IGF-I could not be detected. mRNAs for the IGF type II receptor could be easily demonstrated, whereas those for the insulin- and IGF type I receptor were hardly detectable. In contrast to patient serum tumour cyst fluid strongly stimulated the insulin receptor in vitro. The present study suggests an important role of the simultaneous production of IGF-II and IGFBP-6 in the pathophysiology of tumour-induced hypoglycaemia.