Peptides isolated from cell walls of Medicago truncatula nodules and uninfected root.

The hydroxyproline-rich root nodules of legumes provide a microaerobic niche for symbiotic nitrogen-fixing Rhizobacteria. The contributions of the cell wall and associated structural proteins, particularly the hydroxyproline-rich glycoproteins (HRGPs), are therefore of interest. Our approach involved identification of the protein components by direct chemical analysis of the insoluble wall. Chymotryptic peptide mapping showed a "P3-type" extensin containing the highly arabinosylated Ser-Hyp4-Ser-Hyp-Ser-Hyp4-Tyr3-Lys motif as a major component. Cell wall amino acid analyses and quantitative hydroxyproline arabinoside profiles, predominantly of tri- and tetraarabinosides, confirmed this extensin as the major structural protein in the cell walls of both root nodules and uninfected roots. On the other hand, judging from the Pro, Glu and non-glycosylated Hyp content, the nodule-specific proline-rich glycoproteins, such as the early nodulins (ENOD-PRPs), are present in much lesser amounts. Although we isolated no PRP peptides from nodule cell walls, a single PRP peptide from root cell walls confirmed the presence of a PRP in roots and represented the first direct evidence for a crosslinked PRP in muro. Compared with root cell walls (approximately 7% protein dry weight) nodule cell walls contained significantly more protein (approximately 13% dry weight) with an overall amino acid and peptide composition indicating the presence of structural protein unrelated to the HRGPs.

Molecular cloning and sequencing of cytochrome c' from the phototrophic purple sulfur bacterium Chromatium vinosum.

The gene for cytochrome c' from Chromatium vinosum was cloned from a HindIII-SalI digest of genomic DNA. A 1.4 kbp fragment containing the gene was sequenced in both directions using the Sanger dideoxy method. The cytochrome c' gene codes for a 154-residue peptide, of which the last 131 amino acids match the previously determined sequence of the protein. The remaining 23 residues represent a signal sequence that is cleaved from the polypeptide upon translocation to the periplasmic space. An additional open reading frame on the other strand of the fragment codes for a peptide that contains four regions that are homologous to corresponding regions of the cytochrome b-type subunit of several Ni-Fe hydrogenases.

Nucleotide sequence of the heme subunit of flavocytochrome c from the purple phototrophic bacterium, Chromatium vinosum. A 2.6-kilobase pair DNA fragment contains two multiheme cytochromes, a flavoprotein, and a homolog of human ankyrin.

The gene for the cytochrome subunit of Chromatium vinosum flavocytochrome c (sulfide dehydrogenase) was cloned from an EcoRI digest of chromosomal DNA. The mature cytochrome subunit contains 175 amino acid residues and two heme binding sites in agreement with the previously reported amino acid sequence. There is also a signal peptide of 25 residues, which apparently directs the protein to the periplasmic space. There are two open reading frames upstream of the heme subunit gene, which encode a tetraheme cytochrome c and a homolog of human ankyrin. The gene for the flavoprotein subunit of flavocytochrome c is in frame 15 nucleotides downstream of the stop codon for the cytochrome gene. Messenger RNA was isolated from malate grown cells. The transcript is approximately 3 kilobases in size and does not hybridize with a probe containing the tetraheme cytochrome gene and part of the ankyrin homolog gene. The heme subunit and flavoprotein subunit genes thus appear to form an operon. The flavoprotein subunit has a 30-residue signal peptide. The clone ends 95 amino acids into the N-terminal sequence of the mature flavoprotein subunit (which should contain about 400 residues). The apparently periplasmic location of flavocytochrome c has important consequences for the presumed function as a sulfide dehydrogenase, because sulfur, which is the product of oxidation, is stored in the cytoplasm. Our results on the location of the enzyme are incompatible with this function.

Covalent structure of the diheme cytochrome subunit and amino-terminal sequence of the flavoprotein subunit of flavocytochrome c from Chromatium vinosum.

The complete sequence of the 21-kDa cytochrome subunit of the flavocytochrome c (FC) from the purple phototrophic bacterium Chromatium vinosum has been determined to be as follows: EPTAEMLTNNCAGCHG THGNSVGPASPSIAQMDPMVFVEVMEGFKSGEIAS TIMGRIAKGYSTADFEKMAGYFKQQTYQPAKQSF DTALADTGAKLHDKYCEKCHVEGGKPLADEEDY HILAGQWTPYLQYAMSDFREERRPMEKKMASKL RELLKAEGDAGLDALFAFYASQQ. The sequence is the first example of a diheme cytochrome in a flavocytochrome complex. Although the locations of the heme binding sites and the heme ligands suggest that the cytochrome subunit is the result of gene doubling of a type I cytochrome c, as found with Azotobacter cytochrome c4, the extremely low similarity of only 7% between the two halves of the Chromatium FC heme subunit rather suggests that gene fusion is at the evolutionary origin of this cytochrome. The two halves also require a single residue internal deletion for alignment. The first half of the Chromatium FC heme subunit is 39% similar to the monoheme subunit of the FC from the green phototrophic bacterium Chlorobium thiosulfatophilum, but the second half is only 9% similar to the Chlorobium subunit. The N-terminal sequence of the Chromatium FC flavin subunit was determined up to residue 41 as AGRKVVVVGGGTGGATAAKYIKLADPSIEVTLIEP NTKYYT. It shows more similarity to the Chlorobium FC flavin subunit (60%) than do the two heme subunits. The N terminus of the flavin subunit is homologous to a number of flavoproteins, including succinate dehydrogenase, glutathione reductase, and monamine oxidase. There is no obvious homology to the Pseudomonas putida FC flavin subunit, which suggests that the two types of flavocytochrome c arose by convergent evolution. This is consistent with the dissimilar enzyme activities of FC as sulfide dehydrogenase in the phototrophic bacteria and as p-cresol methylhydroxylase in Pseudomonas. We also present a sequence "fingerprint" pattern for the recognition of FAD-binding proteins which is an extended version of the consensus sequence previously presented (Wierenga, R. K., Terpstra, P., and Hol, W. G. J. (1986) J. Mol. Biol. 187, 101-107) for nucleotide binding sites.

Localisation of a mutation producing autosomal dominant polycystic kidney disease without renal failure.

A four generation Finnish family was identified with atypical features of adult polycystic kidney disease. All members of the extended pedigree were asymptomatic and none had developed renal failure. Previous studies have shown close linkage between the adult polycystic kidney disease locus and the alpha chain of human haemoglobin on chromosome 16, but these studies were carried out on families manifesting 'typical' clinical features of the disease. In order to determine whether the atypical clinical features observed in this Finnish family were produced by a mutation at the same or a second locus, linkage studies were carried out using a highly polymorphic DNA marker from the alpha globin cluster. Here we show that the mutation producing the disease in this Finnish family is also closely linked to alpha globin.

Prenatal diagnosis of autosomal dominant polycystic kidney disease with a DNA probe.

A highly polymorphic DNA probe genetically linked to the locus of autosomal dominant polycystic kidney disease was used in linkage studies for prenatal diagnosis in a nine-week fetus at risk for the disease. The fetus was judged to have inherited the polycystic kidney disease mutation, and this was confirmed by microscopic examination of the fetal kidneys at necropsy.

[Studies on the cervical spine: evaluation of the muscular strength and range of active movements]. / Badania nad odcinkiem szyjnym kregoslupa: ocena sil miesniowych i zakresu ruchów czynnych.

Results of examinations of active motions and muscle force of normal cervical spine in 38 metal-industry manual workers have been presented. Comparative values have been obtained which may promote evaluation of pathological changes within the cervical spine. Some correlations have been found between the range of active motions and the force in the following motions: backward bending, head bending to the right, head bending to the left. It has been also observed that the difference between the mean force during forward bending and the mean force during backward bending is statistically significant at the significance level of 0.95.