Evidence that the Plant Host Synthesizes the Heme Moiety of Leghemoglobin in Root Nodules1

Although it is well established that the plant host encodes and synthesizes the apoprotein for leghemoglobin in root nodules, the source of the heme moiety has been uncertain. We recently found that the transcript for coproporphyrinogen III oxidase, one of the later enzymes of heme synthesis, is highly elevated in soybean (Glycine max L.) nodules compared with roots. In this study we measured enzyme activity and carried out western-blot analysis and in situ hybridization of mRNA to investigate the levels during nodulation of the plant-specific coproporphyrinogen oxidase and four other enzymes of the pathway in both soybean and pea (Pisum sativum L.). We compared them with the activity found in leaves and uninfected roots. Our results demonstrate that all of these enzymes are elevated in the infected cells of nodules. Because these are the same cells that express apoleghemoglobin, the data strongly support a role for the plant in the synthesis of the heme moiety of leghemoglobin.

Analysis of genetic instability during mammary tumor progression using a novel selection-based assay for in vivo mutations in a bacteriophage lambda transgene target.

Genetic instability is thought to be responsible for the numerous genotypic changes that occur during neoplastic transformation and metastatic progression. To explore the role of genetic instability at the level of point mutations during mammary tumor development and malignant progression, we combined transgenic mouse models of mutagenesis detection and oncogenesis. Bitransgenic mice were generated that carried both a bacteriophage lambda transgene to assay mutagenesis and a polyomavirus middle T oncogene, mammary gland-targeted expression of which led to metastatic mammary adenocarcinomas. We developed a novel assay for the detection of mutations in the lambda transgene that selects for phage containing forward mutations only in the lambda cII gene, using an hfl- bacterial host. In addition to the relative ease of direct selection, the sensitivity of this assay for both spontaneous and chemically induced mutations was comparable to the widely used mutational target gene, lambda lacI, making the cII assay an attractive alternative for mutant phage recovery for any lambda-based mouse mutagenesis assay system. The frequencies of lambda cII- mutants were not significantly different in normal mammary epithelium, primary mammary adenocarcinomas, and pulmonary metastases. The cII mutational spectra in these tissues consisted mostly of G/C-->A/T transitions, a large fraction of which occurred at CpG dinucleotides. These data suggest that, in this middle T oncogene model of mammary tumor progression, a significant increase in mutagenesis is not required for tumor development or for metastatic progression.