Segments of the POU domain influence one another''s DNA-binding specificity.

The ubiquitously expressed mammalian POU-domain protein Oct-1 specifically recognizes two classes of cis-acting regulatory elements that bear little sequence similarity, the octamer motif ATGCAAAT and the TAATGARAT motif. The related pituitary-specific POU protein Pit-1 also recognizes these two motifs but, unlike Oct-1, binds preferentially to the TAATGARAT motif. Yet in our assay, Pit-1 still binds octamer elements better than does the octamer motif-binding protein Oct-3. The POU domain is responsible for recognizing these diverse regulatory sequences through multiple DNA contacts that include the two POU subdomains, the POU-specific region, and the POU homeodomain. The DNA-binding properties of 10 chimeric POU domains, in which different POU-domain segments are derived from either Oct-1 or Pit-1, reveal a high degree of structural plasticity; these hybrid proteins all bind DNA well and frequently bind particular sites better than does either of the parental POU domains. In these chimeric POU domains, the POU-specific A and B boxes and the hypervariable POU linker can influence DNA-binding specificity. The surprising result is that the influence a particular segment has on DNA-binding specificity can be greatly affected by the origin of other segments of the POU domain and the sequence of the binding site. Thus, the broad but selective DNA-binding specificity of Oct-1 is conferred both by multiple DNA contacts and by dynamic interactions within the DNA-bound POU domain.     

Induction and modification of dinitrogenase reductase in the unicellular cyanobacterium Synechocystis BO 8402

Oxygen is an important regulatory factor of nitrogenase induced in a unicellular cyanobacterium, Synechocystis BO 8402, during nitrogen starvation. Synthesis of the enzyme is limited by the efficiency of the cells to remove oxygen by respiration, supported by hydrogenases and, in the light, by inhibition of photosynthesis. With a polyclonal antibody against dinitrogenase reductase (the Fe protein of nitrogenase) a single polypeptide is detected, indicative of an active dimeric enzyme in dense cell suspensions. Inhibition of nitrogenase by addition of oxygen is accompanied by the appearance of a second polypeptide of the Fe protein having a 1.5 kDa higher molecular weight. This disappears upon removal of oxygen from the gas phase while nitrogenase activity is restored. No protein synthesis is required indicating that a fraction of the existing polypeptides is reversibly modified in response to oxygen. After induction of nitrogenase activity in dilute culture suspensions, both forms of the Fe-protein are found in variable amounts possibly due to oxygen contamination during the experiment.Abbreviations CAM chloramphenicol - Chl chlorophyll a - CHO carbohydrates - DCMU 3,4-dichlorophenyl-1,1-dimethylurea (diuron) - kDa kilodalton - SDS sodium dodecylsulphate     

Molecular genetic analysis of 67 patients with duchenne/becker muscular dystrophy

Summary A total of 56 Duchenne muscular dystrophy (DMD) patients and 11 Becker muscular dystrophy (BMD) patients was analyzed by extended multiplex amplification of the DMD/BMD gene; deletions were found in 60% of these patients. The data obtained were used to test the frameshift hypothesis and to compare the distribution of familial versus isolated cases. A significant correlation was found between deletions and isolated cases. Additional experiments were performed in order to determine the deletion breakpoints more precisely. These data are a prerequisite for carrier analysis in the respective families by detection or exclusion of aberrant cDNA fragments derived from ectopic lymphocyte RNA. This diagnostic technique is illustrated by 5 examples.     

Retino-fugal projections in congenitally monophthalmic fish and anurans

Summary Intra-ocular deposition of horseradish peroxidase was used to visualize optic tract projections in normal and congenitally monophthalmic catfish and Xenopus. In neither species was evidence for an increased ipsilateral visual component found in congenitally one-eyed specimens. This indicates that competition between axons from both eyes is not an important mechanism for fiber distribution in the chiasm during ontogeny. Furthermore, it suggests that enhanced ipsilateral components, previously noted in unilaterally enucleated fish and anurans, are caused by debris of degenerated axons.     

Characterization of a monoclonal antibody to a conserved epitope on human seminal vesicle-specific peptides: a novel probe/marker system for semen identification

A novel sperm-coating antigen from the human seminal vesicles was discovered. We identified a monoclonal antibody MHS-5, recognizing an epitope with characteristics of a forensic semen marker: conservation in all vasectomized or normal semen samples tested (421); absence in all human tissues or biological fluids other than semen; and immunolocalization on the surface of ejaculated sperm. Western blots of ejaculates allowed to liquefy for 5 min demonstrated the MHS-5 epitope to be located on peptides of a wide range of molecular masses from 69 to 8 kDa. After 15 h of semen liquefaction, immunoreaction peptides of higher molecular mass were undetectable in semen, while peptides of lower molecular mass from 8 to 21 kDa retained antigenicity. Three peptides of 10, 11.9, and 13.7 kDa were the most immunoreactive species in semen liquified for 15 h. Using the MHS-5 monoclonal, an enzyme-linked immunosorbent assay (ELISA) was developed sensitive to 1 ng of seminal protein. This assay showed that the MHS-5 antigen was undetectable in semen of common domestic animals and monkeys but was present in chimpanzee, gorilla, and orangutan semen. ELISA of homogenates from human organs and reproductive tissues demonstrated the antigen only in samples of seminal vesicles. Epididymal sperm obtained at vasovasostomy lacked the MHS-5 epitope, a fact that, together with immunolocalization on ejaculated sperm, demonstrated that the MHS-5 antigen functions as a "sperm-coating antigen." The MHS-5 monoclonal detected semen in sexual-assault evidence obtained six months previously and in mixtures of semen with vaginal or cervical fluid. Assay systems employing the MHS-5 monoclonal may be useful for identification of semen in sexual-assault casework. The MHS-5 epitope resides on novel seminal vesicle-specific peptides whose functions, aside from sperm coating, are uncharacterized.     

Age changes of skull dimensions

At cranial level, external apposition during ageing has been postulated by some authors. In longitudinal studies, a gradual increase of cranial diameters has been shown by cephalometry (Kendrick et al. 1967) or by lateral radiography (Israel 1968, 1970). However, these results are contested at methodological level by other longitudinal studies (Tallgren 1974). It is the aim of this study to analyse, in a cross-sectional sample, the effects of senescence on several cephalic dimensions. A series of skulls of known age and sex has been selected for this purpose.     

Molecular cloning of a pea mRNA encoding an early light induced,nuclear coded chloroplast protein

Summary cDNA clones were isolated for a chloroplast protein, the mRNA of which is induced to maximum levels within 2–4 h after onset of illumination in five day old, etiolated pea seedlings. The cDNA library was constructed from poly(A)⁺-mRNA which was isolated from 4 h illuminated seedlings. The extremely short induction period of the early light induced protein(ELIP)-mRNA established the basis of our screening procedure. Colony hybridization experiments were performed with³²P-labelled cDNA probes, synthesized from RNA of seedlings which had been exposed to different programs of illumination. Plasmid DNAs were isolated from colonies showing strong hybridization signals exclusively with cDNA corresponding to the 4 h-mRNA. Hybrid released translation of preselected plasmids p 17/C2 and p17/C4 revealed a peptide of M_r 24 000. After posttranslational importin vitro, the processed product of M_r 17 000 appears in the chloroplast. Using these clones, the expression of the ELIP-mRNA was investigated by DOT-hybridization. The ELIP-mRNA reaches maximum levels within 2–4 hours after onset of illumination. Our results correspond precisely to thein vivo characteristics and indicate positive identification of the sought clones.