In plants, glutamine synthetase (GS) is the enzyme primarily responsible for the assimilation of ammonia into organic nitrogen. In Phaseolus vulgaris a number of isoenzymic forms of GS are found, each of which consists of eight subunits of mol. wt 41 000‐45 000. The GS subunits of P. vulgaris have previously been shown to be encoded by a small multigene family and a partial cDNA clone for a nodule‐specific GS subunit has been obtained. We report here the isolation and nucleotide sequencing of two essentially full‐length GS cDNA clones (pR‐1 and pR‐2) from a root cDNA library and the deduced amino acid sequences of the corresponding GS subunits (355 amino acid residues each). The coding sequences of pR‐1 and pR‐2 are closely related (80% nucleotide homology, 88% amino acid homology), but their 5′‐ and 3′‐untranslated regions have diverged almost completely. Both pR‐1 and pR‐2 are related to, but distinct from, the nodule GS clone, pcPvNGS‐01 (or pN‐1). Hybridization to genomic Southern blots showed that the three GS mRNAs are encoded by three seperate genes and indicated the existence of a fourth class of GS gene. An S1 nuclease protection assay demonstrated the presence of R‐1 and R‐2 mRNA in both roots and leaves and confirmed that expression of the N‐1 gene is nodule‐specific. Expression of the R‐1 and R‐2 genes in the roots did not change significantly during nodulation. However, only the R‐1 gene is expressed in the nodules themselves, indicating that the R‐2 gene is specifically repressed during nodule development.

The in vivo intraovarian synthesis of insulin-like growth factor-I (IGF-I) has been studied in the rat by Northern blot, dot blot hybridization, and in situ hybridization histochemistry. Ovarian IGF-I mRNA transcript sizes (7.0 kilobases (kb), 1.6 kb, and a group from 0.4-0.9 kb) were similar to those in liver and other tissues. The proportion of ovarian IGF-I to tubulin messenger RNA (mRNA) was increased to 176% of control values by treatment with diethylstilbestrol, while the ratio in liver was decreased to 64.4%. In situ hybridization identifies the major in vivo site of IGF-I synthesis in the ovary as the granulosa cells of developing follicles. IGF-I mRNA was present in the granulosa cells of developing preantral and antral follicles, but was not seen in atretic follicles or corpora lutea. In preovulatory follicles high levels of IGF-I mRNA were confined to the antral cell layers and to the cells of the cumulus oophorus. High levels of tubulin gene expression within follicles were seen in a similar distribution to that for IGF-I but 80-90% of corpora lutea also strongly expressed the tubulin gene. Interstitial cells, including thecal cells, express the tubulin gene at low levels but do not express the IGF-I gene. The distribution of IGF-I mRNA is the same as that previously observed for mitotically active granulosa cells, and therefore offers strong support for the view that IGF-I in the ovary acts by an autocrine-paracrine mechanism to promote granulosa cell replication.

The gln-gamma gene, which specifies the gamma subunit of glutamine synthetase in Phaseolus vulgaris L., has been isolated and the regulatory properties of its promoter region analyzed in transgenic Lotus corniculatus plants. A 2-kilobase fragment from the 5'-flanking region of gln-gamma conferred a strongly nodule-enhanced pattern of expression on the beta-glucuronidase reporter gene. Parallel studies on the promoter of another glutamine synthetase gene (gln-beta) showed that a 1.7-kilobase fragment directed 20-fold to 140-fold higher levels of beta-glucuronidase expression in roots than in shoots. Histochemical localization of beta-glucuronidase activity in nodules of the transgenic plants indicated that the chimeric gln-gamma gene was expressed specifically in the rhizobially infected cells; expression of the gln-beta construct was detected in both cortical and infected regions of young nodules, and became restricted to the vascular tissue as the nodule matured. We conclude that gln-beta and gln-gamma genes are differentially expressed both temporally and spatially in plant development and that the cis-acting regulatory elements responsible for conferring these contrasting expression patterns are located within a 2-kilobase region upstream of their coding sequences.

The Ehlers-Danlos syndrome (EDS) is a heterogeneous group of inherited connective tissue disorders characterised by skin hyperextensibility, joint hypermobility, easy bruising, and cutaneous fragility. Nine discrete clinical subtypes have been classified. We have investigated the molecular defect in a patient with clinical features of Ehlers-Danlos syndromes types I/II and VII. Electron microscopy of skin tissue indicated abnormal collagen fibrillogenesis with longitudinal sections showing a marked disruption of fibril packing giving very irregular outlines to transverse sections. Analysis of the collagens produced by cultured fibroblasts showed that the type V collagen had a population of al(V) chains shorter than normal. Peptide mapping suggested a deletion within the triple helical domain. RT-PCR amplification of mRNA covering the whole of this domain of COL5A1 showed a deletion of 54 bp. Although six Gly-X-Y triplets were lost, the essential triplet amino acid sequence and C-propeptide structure were maintained allowing mutant protein chains to be incorporated into triple helices. Genomic DNA analysis identified a de novo G+3-+T transversion in a 5' splice site of one COL5A1 allele. This mutation is analogous to mutations causing exon skipping in the major collagen genes, COLlA1, COL1A2, and COL3A1, identified in several cases of osteogenesis imperfecta and EDS type IV. These observations support the hypothesis that type V, although quantitatively a minor collagen, has a critical role in the formation of the fibrillar collagen matrix. (Burrows et al, in press, Burrows et al, in preparation).Type V collagen is a low abundance fibrillar collagen with a wide distribution. It occurs in such diverse tissues as fetal membranes, placenta, skin, bone, cartilage, tendon, cornea, synovial membrane, blood vessel walls, liver, and lung. Three distinct cx chains have been Here we present the biochemical and molecular characterisation of a type V collagen defect in a young woman with atypical EDS II. She produces both normal and shortened ocl(V) chains with some carrying a deletion in the triple helical domain as a result of skipping a 54 bp exon in the mRNA transcripts because of a point mutation in one COL5A1 allele. We believe this represents the first characterisation of a naturally occurring structural mutation in a human type V collagen gene. Materials and methods CASE REPORTClinical examination of the proband, a 24 year old woman, showed generalised skin fragility with extensive scarring of the forehead, shins, and knees and scattered bruising on the arms and legs. There was a marked generalised joint laxity with severe premature bilateral hallux valgus and diamond shaped feet. She was short (155 cm) with a mild thoracic kyphoscoliosis,

The gln-y gene, encoding the y subunit of glutamine synthetase in French bean (Phaseolus vugaris), is strongly induced during nodule development. We have determined the nucleotide sequence of a 1.3-kilobase region at its 5' end and have identified severa1 sequences common to the promoter regions of late nodulin genes from other legume species. The fi'-flanking region was analyzed for sequence-specific interactions with nuclear factors from French bean. A factor from nodules (PNF-1) was identified that binds to multiple sites between -860 and -154, and a related but distinct factor (PRF-1) was detected in extracts from uninfected roots. PNF-1 and PRF-1 bound strongly to a synthetic oligonucleotide containing the sequence of an A/T-rich 21-base pair imperfect repeat found at positions -516 and -466. The same factors also had a high affinity for a protein binding site from a soybean leghemoglobin gene and appeared to be closely related to the soybean nodule factor NAT2, which binds to A/Trich sequences in the lbcs and nodulin 23 genes [Jacobsen et al. (1990). Plant Cell2, 85-94]. Comparison of NAT2/ PNF-1 binding sites from a variety of nodulin genes revealed the conservation of the short consensus core motif TATTTWAT, and evidence was obtained that this sequence is important for protein recognition. Cross-recognition by PNF-1 of a protein binding site in a soybean seed protein gene points to the existence of a ubiquitous family of factors with related binding affinities. Our data suggest that PNF-1 and PRF-1 belong to an evolutionarily conserved group of nuclear factors that interact with specific A/T-rich sequences in a diverse set of plant genes. We consider the possible role of these factors in coregulating the expression of g h -r and other late nodulin genes.